Blinded by smoke: Wildfire smoke exposure and eye irritation in Australian wildland firefighters.

PURPOSE: Wildfire occurrence is increasing worldwide, putting firefighters and general public at increased risk of eye injuries from smoke exposure. This study explored ocular symptoms and use of protective eyewear amongst wildland firefighters in Australia. METHODS: Australian wildland firefighters were invited to complete an online survey about the occurrence of eye irritation, use of protective eyewear and behaviours associated with occupational smoke exposure. Responses were analysed using logistic regression and qualitative inductive content analysis. RESULTS: 338 wildland firefighters completed the survey. Eye irritation was reported by 90% of firefighters at least sometimes during work and by 70% after work. Frequency of eye irritation was greater amongst females than males (OR 2.01, CI 1.22 - 3.31, p<0.05). Protective eyewear was used often or always by 67% of firefighters on the fireground, however 55% had to remove their protective eyewear due to sweat, fogging or another reason. Goggles were more likely to be removed compared to sunglasses and safety glasses (OR 4.28, CI 2.75 - 6.68, p<0.001). Firefighters reported that, at times smoke exposure necessitated eye closure and impaired vision on the fireground. Firefighters also reported that protective eyewear helped to reduce eye symptoms, but its consistent use on the fireground was difficult. The severity and recovery from eye symptoms varied between participants. CONCLUSION: Australian wildland firefighters frequently experience eye irritation from smoke exposure, and this can affect operational capabilities. These findings can support the development of evidence-based strategies to help protect and aid recovery of the eye surface following smoke exposure.

Epithelial Immune Cell Response to Initial Soft Contact Lens Wear in the Human Corneal and Conjunctival Epithelium.

Purpose: The purpose of this study was to assess the immediate ocular immune response to soft contact lens (CL) wear by examining presumed epithelial immune cell (EIC) density and morphology at the central, peripheral, limbal cornea, and conjunctiva. Methods: Fifty-four participants naïve to CL wear (mean age = 24.8 ± 9.8 years, 44% female participants), were examined using in vivo confocal microscopy at baseline and after 2 hours of CL wear (1-Day ACUVUE MOIST). Images were captured at the central, temporal far peripheral and limbal cornea, and bulbar conjunctiva. EIC density was counted manually and morphology was graded. Differences in EIC parameters pre- and post-CL wear were examined using a generalized estimating equation model with appropriate post hoc analyses. Results: After 2 hours of soft CL wear, there was a significant increase in EIC density in all regions other than the central cornea (all P < 0.001). Cell body size was significantly larger, and a higher proportion of participants exhibited EIC with long dendrites after lens wear at the central and peripheral cornea (both P < 0.001). There was a significant increase in the number of participants displaying EIC with thick dendrites at the peripheral (P = 0.04) and limbal cornea (P < 0.001) after lens wear. Conclusions: EICs were primarily recruited to the peripheral regions, whereas the central cornea shows no significant recruitment after short-term CL wear. Both central and peripheral corneas exhibited an enhanced antigen capture capacity, whereas migratory capacity was increased in the peripheral corneal regions suggesting EIC activation following a short period of CL wear.

Dendritic Cell Density and Morphology Can Be Used to Differentiate Vernal Keratoconjunctivitis from Allergic Conjunctivitis.

The aim of the study was to compare the distribution of corneal and conjunctival epithelial dendritic cells (DCs) in vernal keratoconjunctivitis (VKC), allergic conjunctivitis (AC), and non-allergic controls to examine if the allergy type causes differences in immune cell activation. The prospective study included 60 participants: 20 with VKC, 20 with AC, and 20 non-allergic controls. In vivo confocal microscopy was performed on the right eye. The locations scanned included the corneal centre, inferior whorl, corneal periphery, corneal limbus, and bulbar conjunctiva. The DCs were counted manually, and their morphology was assessed for the largest cell body size, the presence of dendrites, and the presence of long and thick dendrites. The DC density was higher in VKC and AC compared to non-allergic group at all locations (p ≤ 0.01) except at the inferior whorl. The DC density in VKC participants was significantly higher than in AC at the limbus (p < 0.001) but not at other locations. Both the AC and the VKC group had larger DC bodies at the corneal periphery and limbus compared to the non-allergic group (p ≤ 0.03). The study found a higher proportion of participants with DCs exhibiting long dendrites at both the corneal periphery in AC (p = 0.01) and at the corneal centre, periphery, and limbus in VKC, compared to the non-allergic group (p ≤ 0.001). In conclusion, a higher DC density at the limbus may be a marker of more severe VKC. DCs with larger cell bodies and a greater proportion of participants with DCs displaying long dendrites can be potential markers to differentiate allergy from non-allergy, and more severe forms of allergy from milder forms.

Culture of primary human meibomian gland cells from surgically excised eyelid tissue.

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 µM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

Effects of elevated serum estrogen on dry eye in women undergoing in vitro fertilisation.

PURPOSE: Sex hormones impact inflammatory and immune-mediated diseases. During IVF (in vitro fertilisation) treatment, circulating estrogen levels increase dramatically (10-50x) alongside changes in other hormones. This study examined changes in dry eye with IVF and its relationship with sex hormones. METHODS: A two visit study was conducted on first day of menstruation when estrogen levels are lowest (baseline visit), and on day 9-11 (peak estrogen visit (PO)) of IVF. Symptoms of dry eye and ocular pain and signs of dry eye were examined. Serum hormone levels were assessed using mass spectrometry and immunoassay. Changes in signs and symptoms and associations were explored. Hierarchical multiple regression analysis assessed factors contributing to signs and symptoms. RESULTS: 40 women (36.2 ± 4.0 years) completed the study. Baseline and PO oestradiol (E2) levels were 28.9 pg/ml (20) (median (IQR)); 1360 pg/ml (1276) respectively. Ocular pain and dry eye symptoms worsened (p = 0.02 and p < 0.01) and tear break up and tear secretion values decreased (p = 0.005 and 0.01) at PO. Higher E2 and lower luteinizing hormone (LH) were associated with worsening of dry eye symptoms (ρ = 0.34 p = 0.03, ρ = -0.49 p = 0.001). Reduction in LH and increase in progesterone (P4) were associated with increased ocular pain (ρ = 0.45, p = 0.004 and ρ = 0.39, p = 0.01). Dry eye symptoms were predicted by LH and tear break up (p = 0.02; R2 = 0.18). CONCLUSIONS: IVF treatment resulted in significantly increased ocular symptoms and tear film alterations although these changes were not clinically significant. Dry eye signs and symptoms were poorly predicted by hormone levels.

Increased dendritic cell density and altered morphology in allergic conjunctivitis.

BACKGROUND: Corneal and conjunctival epithelial dendritic cells (DC) have an established role in vernal keratoconjunctivitis, however, their role in more prevalent forms of allergic eye disease remains unclear. This study evaluated corneal and conjunctival epithelial DC density, morphology, and distribution observed using in vivo confocal microscopy (IVCM) in allergic conjunctivitis. METHODS: In this prospective, observational study, 66 participants (mean age 36.6 ± 12.0 years, 56% female): 33 with allergic conjunctivitis and 33 controls were recruited. IVCM was performed at the corneal centre, inferior whorl, corneal periphery, corneal limbus, and temporal bulbar conjunctiva. DC were counted and their morphology was assessed as follows: largest cell body size, presence of dendrites, and presence of long and thick dendrites. Mixed model analysis (DC density) and non-parametric tests (DC morphology) were used. RESULTS: DC density was higher in allergic participants at all locations (p ≤ 0.01), (corneal centre median (IQR) 21.9 (8.7-50.9) cells/mm2 vs 13.1 (2.8-22.8) cells/mm2; periphery 37.5 (15.6-67.2) cells/mm2 vs 20 (9.4-32.5) cells/mm2; limbus 75 (60-120) cells/mm2 vs 58.1 (44.4-66.2) cells/mm2; conjunctiva 10 (0-54.4) cells/mm2 vs 0.6 (0-5.6) cells/mm2, but not at the inferior whorl 21.9 (6.2-34.4) cells/mm2 vs 12.5 (1.9-37.5) cells/mm2, p = 0.20. At the corneal centre, allergic participants had larger DC bodies (p = 0.02), a higher proportion of DC with dendrites (p = 0.02) and long dendrites (p = 0.003) compared to controls. CONCLUSIONS: Corneal and conjunctival DC density was increased, and morphology altered in allergic conjunctivitis. These findings imply that the ocular surface immune response was upregulated and support an increased antigen-capture capacity of DC in allergic conjunctivitis.

Dendritiform immune cells with reduced antigen-capture capacity persist in the cornea during the asymptomatic phase of allergic conjunctivitis.

BACKGROUND: Increased density and altered morphology of dendritic cells (DC) in the cornea and conjunctiva occur during active allergic conjunctivitis. This study investigated whether inflammation (characterised by altered DC density and morphology) persists during the symptom-free phase of allergic conjunctivitis. METHODS: Twenty participants (age 43.3 ± 14.3 years, 55% female) assessed during their active (symptomatic) phase of allergic conjunctivitis were re-examined during the asymptomatic phase. Ocular allergy symptoms and signs were evaluated during both phases, and five ocular surface locations (corneal centre, inferior whorl, corneal periphery, corneal limbus, and bulbar conjunctiva) were examined using in vivo confocal microscopy (HRT III). DC were counted manually, and their morphology was assessed for cell body size, presence of dendrites, presence of long dendrites and presence of thick dendrites using a grading system. Mixed model analysis (DC density) and non-parametric tests (DC morphology) were used to examine differences between phases. RESULTS: DC density at corneal locations did not change between the active and asymptomatic phases (p ≥ 0.22). However, corneal DC body size was smaller and fewer DC presented with long dendrites during the asymptomatic phase (p ≤ 0.02). In contrast, at the bulbar conjunctiva, DC density was reduced during the asymptomatic phase compared to the active phase (p = 0.01), but there were no changes in DC morphology. CONCLUSIONS: Dendritiform immune cell numbers persist in the cornea during the symptom-free phase of allergic conjunctivitis, whereas conjunctival DC appear to return to a baseline state. The morphology of these persisting corneal DC suggests their antigen-capture capacity is reduced during the asymptomatic phase.

Blink Rate Measured In Situ Decreases While Reading From Printed Text or Digital Devices, Regardless of Task Duration, Difficulty, or Viewing Distance.

Purpose: To compare blinking measured in situ during various tasks and examine relationships with ocular surface symptoms. The day-to-day repeatability of the blink rate and interblink interval was assessed. Methods: Twenty-four students (28.6 ± 6.3 years; 8 male and 16 female) completed six reading tasks (printed text, laptop, TV, smartphone, smartphone at 50% brightness, smartphone with complex text), and two nonreading tasks (conversation, walking) in a randomized cross-over study. Ocular surface symptoms and clinical signs were assessed. The blink rate and interblink interval were measured using a wearable eye tracking headset. Blink parameters were compared across tasks and time (linear mixed model and post hoc comparisons with Bonferroni correction). Associations between blinking, symptoms, ocular surface, and clinical signs were assessed (Spearman's correlation). The smartphone reading task was completed twice to determine the coefficient of repeatability. Results: The blink rate was lower (mean 10.7 ± 9.7 blinks/min) and the interblink interval longer (mean 9.6 ± 8.7 seconds) during all reading tasks compared with conversation (mean 32.4 ± 12.4 blinks/min; 1.5 ± 0.6 seconds) and walking (mean 31.3 ± 15.5 blinks/min; 1.9 ± 1.3s) (P < 0.001). There were no significant differences in blink parameters between any of the reading tasks or between conversation and walking. Changes in blinking occurred within 1 minute of starting the task. No associations were evident between blink rate or interblink interval and ocular surface symptoms or signs. The coefficient of repeatability was ±12.4 blinks/min for blink rate and ±18.8 seconds for interblink interval. Conclusions: Spontaneous blinking can be measured reliably in situ. The blink rate was decreased and the interblink interval increased during reading compared with conversation and walking. Changes in blinking were immediate, sustained, and not associated with ocular surface symptoms or signs.

Diurnal changes and topographical distribution of ocular surface epithelial dendritic cells in humans, and repeatability of density and morphology assessment.

PURPOSE: Dendritic cells (DC) play a crucial role in ocular surface defence. DC can be visualised in vivo by confocal microscopy but have not yet been fully characterised in humans. This study investigated the diurnal variation, topographical distribution and repeatability of DC density and morphology measurements. METHODS: In vivo confocal microscopy (IVCM) was conducted on 20 healthy participants (mean age 32.7 ± 6.4 years, 50% female) at baseline and repeated after 30 minutes, 2, 6 and 24 h. Images were captured at the corneal centre, inferior whorl, corneal periphery, limbus and bulbar conjunctiva. DC were counted manually, and their morphology was assessed for cell body size, presence of dendrites, and presence of long and thick dendrites. Mixed-model analysis, non-parametric analyses, Bland and Altman plots, coefficient of repeatability (CoR) and kappa were used. RESULTS: There were no significant changes in DC density (p ≥ 0.74) or morphology (p > 0.07) at any location over the 24-h period. The highest DC density was observed at the corneal limbus followed by the peripheral cornea (p < 0.001), with the lowest density at the corneal centre, inferior whorl and bulbar conjunctiva. Most DC at the corneal periphery, limbus and bulbar conjunctiva had larger cell bodies compared with the corneal centre (p ≤ 0.01), and the presence of long dendrites was observed mostly at non-central locations. Day-to-day CoR for DC density ranged from ±28.1 cells/mm2 at the corneal centre to ±56.4 cells/mm2 at the limbus. Day-to-day agreement of DC morphology determined by kappa ranged from 0.5 to 0.95 for cell body size, 0.60 to 0.95 for presence of dendrites, and 0.55 to 0.80 for the presence of long dendrites at various locations. CONCLUSIONS: No diurnal changes are apparent in corneal or conjunctival DC. Substantial topographical differences exist in DC density and morphology. IVCM provides good repeatability of DC density and acceptable agreement of DC morphology.

Smartphone gaming induces dry eye symptoms and reduces blinking in school-aged children.

PURPOSE: Smartphone use by children is rising rapidly, but its ocular surface impact is unknown. This study examined the effect of smartphone use on blinking, symptoms, and tear function in children. METHODS: Prospective intervention study where 36 children aged 6-15years (14 M:22 F) played games on a smartphone continuously for one hour. Symptoms (SANDE, IOSS, NRS) and tear film (lipid layer thickness, tear secretion, stability) were assessed before and after gaming. Blink rate and interblink interval were measured in situ using an eye tracking headset, before (during conversation) and continuously throughout gaming. Symptoms and tear film changes were examined using paired t-tests. Changes in blinking throughout one hour were examined using repeated measures ANOVA, post-hoc comparisons with Bonferroni correction. Associations examined using Pearson bivariate correlation. Significance level was 0.05. RESULTS: Symptoms worsened following one hour smartphone gaming (SANDE + 8.2units, p = 0.01; IOSS + 1.3units, p < 0.001; NRS-average +6.3units, p = 0.03; NRS-comfort +7.6units, p = 0.04; NRS-tiredness +10.1units, p = 0.01), but tear film remained unchanged. Blink rate reduced from 20.8 blinks/min to 8.9 blinks/min (p < 0.001) and interblink interval increased from 2.9 s to 8.7 s (p = 0.002) within the first minute of gaming relative to baseline conversation, and this effect remained unchanged throughout one hour of gaming. CONCLUSIONS: Smartphone use in children results in dry eye symptoms and immediate and sustained slowing of blinking, with no change in tear function evident up to one hour. Given the ubiquitous use of smartphones by children, future work should examine whether effects reported herein persist or get worse over a longer term causing cumulative damage to the ocular surface.

Corneal epithelial dendritic cells, tear neuropeptides and corneal nerves continue to be affected more than 12 months after LASIK.

PURPOSE: LASIK causes corneal nerve damage and may affect the neuro-immune crosstalk. This study examined the effects of LASIK on corneal epithelial dendritic cells (CEDC) density and morphology and explored their relationships with corneal nerves and tear neuropeptides. A grading system was developed to assess CEDC morphology. METHODS: Intra- and inter-observer repeatability of the CEDC morphology grading system was established using kappa (κ). In vivo confocal microscope images of the central cornea were captured from 20 participants who had undergone LASIK 12-16 months earlier and 20 controls (age 18-32 years, 55%F). CEDC density was counted manually, and CEDC morphology was assessed using a new grading system. CEDC sub-types (contacting nerves [CEDCc] and not contacting nerves [CEDCnc]) were also assessed. Differences in CEDC density and morphology were examined using mixed models and chi-squared test. Relationships between CEDC and corneal nerve parameters and tear substance P were explored using Spearman's correlation. RESULTS: Excellent intra- and inter-observer repeatability was demonstrated for the grading system (κ = 0.82-0.97). In post-LASIK participants, CEDC density was lower compared with controls (5 [0-34] vs. 21 [7-77] cells/mm2 ; p = 0.01), and the proportion of CEDC with thick dendrites was higher (55%-73% vs. 11%-21%, p < 0.003). Higher tear substance P levels were associated with higher CEDC density (rho = 0.48, p = 0.003). Fewer nerve interconnections were observed in participants in whom CEDC had dendrites (p = 0.03). CEDC sub-types followed a similar pattern to CEDC. CONCLUSIONS: The findings suggest that CEDC may remain altered more than 12 months post-LASIK. The association with substance P suggests a role for CEDC in corneal neurogenic inflammation.

Blinking and normal ocular surface in school-aged children and the effects of age and screen time.

PURPOSE: Baseline ocular surface characteristics in children require investigation. This study characterised blinking and relationships with ocular symptoms, tear film and digital device use. METHODS: 45 children aged 6-15 years (56% female) participated in a cross-sectional study. Ocular surface symptoms (Instant Ocular Symptoms Survey, Dry Eye Questionnaire 5, Symptoms Assessment in Dry Eye, Ocular Surface Disease Index, Ocular Comfort Index and Numerical Rating Scale) and clinical indices (lipid layer thickness, tear secretion and stability, meibomian gland) were assessed. Blink rate and interblink interval were measured in situ using a wearable eye-tracking headset (Pupil Labs GmbH, Germany). Associations between blinking, ocular surface, age, and digital device use (bivariate and partial correlations) and between automated and manually counted blink rate (Bland & Altman) were examined. RESULTS: Mean blink rate and interblink interval were 20.5±10.5 blinks/min and 2.9±1.9 s during conversation. There was no difference between automated and manual blink rate (p=0.78) and no relationship between blinking and digital device use, age or sex. Mean group symptoms were within normal range and not associated with clinical measurements including blinking. Greater tear volume was associated with a faster blink rate (r=0.46, p=0.001) and shorter interblink interval (r=-0.36, p=0.02). Older age was associated with improved tear volume (r=0.37, p=0.01) and stability (r=0.38, p=0.01). CONCLUSIONS: Blinking characterised in situ was not impacted by age or habitual digital device use. A faster blink rate was associated with greater tear volume but not symptoms. Improved tear function was found with age suggesting that the ocular surface continues to develop through childhood.

Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17ß-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.

Smoke and the eyes: A review of the harmful effects of wildfire smoke and air pollution on the ocular surface.

Wildfires are occurring worldwide with greater frequency and intensity. Wildfires, as well as other sources of air pollution including environmental tobacco smoke, household biomass combustion, agricultural burning, and vehicular emissions, release large amounts of toxic substances into the atmosphere. The ocular surface is constantly exposed to the ambient air and is hence vulnerable to damage from air pollutants. This review describes the detrimental effects of wildfire smoke and air pollution on the ocular surface and resultant signs and symptoms. The latest relevant evidence is synthesised and critically evaluated. A mechanism for the pathophysiology of ocular surface damage will be proposed considering the existing literature on respiratory effects of air pollution. Current strategies to reduce human exposure to air pollutants are discussed and specific possible approaches to protect the ocular surface and manage air pollution induced ocular surface damage are suggested. Further avenues of research are suggested to understand how acute and chronic air pollution exposure affects the ocular surface including the short and long-term implications.

Human meibomian gland epithelial cell culture models: Current progress, challenges, and future directions.

The widely used immortalised human meibomian gland epithelia cell (iHMGEC) line has made possible extensive studies of the biology and pathophysiology of meibomian glands (MG). Tissue culture protocols for iHMGEC have been revised and modified to optimise the growth conditions for cell differentiation and lipid accumulation. iHMGEC proliferate in serum-free medium but require serum or other appropriate exogenous factors to differentiate. Several supplements can enhance differentiation and neutral lipid accumulation in iHMGEC grown in serum-containing medium. In serum-free medium, rosiglitazone, a peroxisome proliferator activator receptor-γ (PPARγ) agonist, is reported to induce iHMGEC differentiation, neutral lipid accumulation and expression of key biomarkers of differentiation. iHMGEC cultured in serum-containing medium under hypoxia or with azithromycin increases DNAse 2 activity, a biomarker of terminal differentiation in sebocytes. The production of lipids with composition similar to meibum has not been observed in vitro and this remains a major challenge for iHMGEC culture. Innovative methodologies such as 3D ex vivo culture of MG and generation of MG organoids from stem cells are important for further developing a model that more closely mimics the in vivo biology of human MG and to facilitate the next generation of studies of MG disease and dry eye.

A pilot study investigating the effect of extended contact lens wear on limbal and central corneal morphology.

PURPOSE: To investigate the effect of long-term extended soft contact lens wear on limbal and central corneal cell morphology, and limbal architecture. METHODS: Each participant attended a study visit involving in vivo confocal microscopy of central corneal and limbal epithelium. Scans were graded by five masked graders for three features: central epithelial irregularity, limbal epithelial irregularity and the prominence of palisades of Vogt. The variability of grades between different graders and the difference of grades between extended wearers and daily soft/non-contact lens wearers were assessed. RESULTS: Nineteen participants (9 extended soft contact lens wearers and 10 daily soft/non-contact lens wearers) aged 31-65 years were enrolled in this study. Scans from 37 eyes were included in the analysis. Agreement between graders for each feature was moderate to good with inter class correlation >0.7. While there were no significant differences in central epithelial cell irregularity (p = 0.527) and the prominence of palisade of Vogt (p = 0.182) between extended or daily soft/non-contact lens wearers, limbal epithelial cell irregularity showed a trend with increased irregularity in extended soft contact lens wearers (p = 0.091). CONCLUSIONS: While no differences in limbal cell morphology and structureor central epithelial cell wasfound in thissubjective grading study of extended wearers compared to daily soft/non-contact lens wearers, further studies using a larger sample size or a longitudinal study design are warranted.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

CLEAR - Effect of contact lens materials and designs on the anatomy and physiology of the eye.

This paper outlines changes to the ocular surface caused by contact lenses and their degree of clinical significance. Substantial research and development to improve oxygen permeability of rigid and soft contact lenses has meant that in many countries the issues caused by hypoxia to the ocular surface have largely been negated. The ability of contact lenses to change the axial growth characteristics of the globe is being utilised to help reduce the myopia pandemic and several studies and meta-analyses have shown that wearing orthokeratology lenses or soft multifocal contact lenses can reduce axial length growth (and hence myopia). However, effects on blinking, ptosis, the function of Meibomian glands, fluorescein and lissamine green staining of the conjunctiva and cornea, production of lid-parallel conjunctival folds and lid wiper epitheliopathy have received less research attention. Contact lens wear produces a subclinical inflammatory response manifested by increases in the number of dendritiform cells in the conjunctiva, cornea and limbus. Papillary conjunctivitis is also a complication of all types of contact lenses. Changes to wear schedule (daily disposable from overnight wear) or lens materials (hydrogel from SiHy) can reduce papillary conjunctivitis, but the effect of such changes on dendritic cell migration needs further study. These changes may be associated with decreased comfort but confirmatory studies are needed. Contact lenses can affect the sensitivity of the ocular surface to mechanical stimulation, but whether these changes affect comfort requires further investigation. In conclusion, there have been changes to lens materials, design and wear schedules over the past 20+ years that have improved their safety and seen the development of lenses that can reduce the myopia development. However, several changes to the ocular surface still occur and warrant further research effort in order to optimise the lens wearing experience.

Dry eye symptoms in children: can we reliably measure them?

PURPOSE: Many dry eye questionnaires are available, but these may not be suitable for paediatric eye care. The feasibility of use and repeatability of symptom questionnaires administered to children was examined. METHODS: Participants aged 6-15 years (n = 62; 25M:37F; 40% male) completed six questionnaires twice in random order at a single visit: Symptoms assessment in dry eye (SANDE), ocular surface disease index (OSDI), numerical rating scale (NRS), ocular comfort index (OCI, n = 30), dry eye questionnaire 5 (DEQ-5) and the instant ocular symptoms survey (IOSS). Completion time and need for assistance were recorded and relationships with age examined (Pearson correlation, independent t-test). The number of participants requiring assistance and with which items were compared (linear mixed model, pairwise test). Repeatability (coefficient of repeatability (CoR), limit of agreement, bias) and intraclass correlation coefficient (ICC) were examined. RESULTS: Completion time was ≤2 min for each individual questionnaire. Younger participants took longer to complete (r = -0.43 to -0.60, p ≤ 0.01), and required more assistance (p ≤ 0.001). Forty-eight participants required assistance with at least one questionnaire. Older children (13-15 years) only required assistance with OSDI (p ≤ 0.004) and NRS (p ≤ 0.003). Participants required more assistance with SANDE, OSDI and NRS than with DEQ-5 and IOSS (p ≤ 0.02) and with gritty (OSDI, 77% of participants; OCI, 100%) and foreign body sensation (NRS, 92%) items. CoR was similar for all questionnaires with no evidence of a learning effect (p > 0.05). ICC was moderate to excellent. CONCLUSIONS: Dry eye questionnaires can be used reliably in paediatric eye care; more time and assistance may be required for younger children. The DEQ-5 and IOSS are recommended for use in younger age children.

Ultrasensitive Serum Estradiol Measurement by Liquid Chromatography-Mass Spectrometry in Postmenopausal Women and Mice.

Accurate measurement of very low circulating estradiol (E2) (<5 pg/ml) in postmenopausal women and in mice is essential to investigating sex steroid action in target tissues. However, direct immunoassays are too inaccurate and conventional mass spectrometry-based measurement too insensitive at these serum E2 levels. We report application of an ultrasensitive method using a novel estrogen-selective derivatization in liquid chromatography-mass spectrometry to measure serum E2, with a detection limit of 0.25 pg/ml in small (0.2 ml) serum volumes that can quantify serum E2 in 98% and serum E1 in 100% of healthy postmenopausal women. Aromatase inhibitor (AI) treatment of postmenopausal women with breast cancer further reduces serum E2 by 85% and serum estrone (E1) by 80%. The wide scatter of circulating E2 in AI-treated women suggests that the degree of sustained E2 depletion, now quantifiable, may be an efficacy or safety biomarker of adjuvant AI treatment. This ultrasensitive method can also measure serum E2 in most (65%) female but not in any male mice. Further studies are warranted using this and comparable ultrasensitive liquid chromatography-mass spectrometry estrogen measurements to investigate the relationship of circulating E2 (and E1) in male, postmenopausal female, and childhood health where accurate quantification of serum estrogens was not previously feasible. This will focus on the direct impact of estrogens as well as the indirect effects of androgen aromatization on reproductive, bone, and brain tissues and, notably, the efficacy and safety of AIs in adjuvant breast cancer treatment.