Conjunctival Impression Cytology Samples Research Articles

Secondary Data Analysis of Inflammation-Related mRNAs in Conjunctival Impression Cytology Samples of Aniridia Patients.

Aniridia is a rare corneal disease that is often associated with aniridia-associated keratopathy (AAK). In AAK, the conjunctival tissue crosses the limbal border, forming a corneal pannus that extends into the corneal center. With increasing AAK severity, corneal pannus formation, vascularization, and ocular surface inflammation increase. The purpose of this study was to investigate inflammation-related mRNA expression in conjunctival epithelial cells in AAK and its relationship with AAK severity. Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age-matched and sex-matched healthy control subjects. RNA was extracted, and mRNA analyses were performed using microarray, which was evaluated for inflammatory markers. In the analyzed aniridia subjects, 70 deregulated mRNAs encoding proinflammatory or antiinflammatory cytokines or factors associated with chronic inflammation, including increased IL-1, IL-8, and MIP3A/CCL20 mRNA. The most downregulated mRNA was TIMP3, and the most upregulated mRNA was Protein c-Fos.Of the 70 mRNAs, 14 inflammation-related genes were altered only in the mild AAK forms, whereas only 2 mRNAs were altered only in the severe AAK forms (TLR4 and PPARG). The expression of numerous proinflammatory and antiinflammatory cytokines is deregulated at the ocular surface of aniridia subjects with mild AAK. Thus, early antiinflammatory treatment may prevent or slow down corneal scarring and pannus formation in aniridia subjects.

Protein profiling of conjunctival impression cytology samples of aniridia subjects.

Congenital aniridia is a rare disease, which is in most cases related to PAX6 haploinsufficiency. Aniridia associated keratopathy (AAK) also belongs to ocular signs of congenital aniridia. In AAK, there is corneal epithelial thinning, corneal inflammation, vascularization and scarring. In advanced stage AAK, typically, conjunctival epithelial cells slowly replace the corneal epithelium. Based on previous results we hypothesize that alterations of the conjunctival cells in congenital aniridia may also support the corneal conjunctivalization process. The aim of this study was to identify deregulated proteins in conjunctival impression cytology samples of congenital aniridia subjects. Conjunctival impression cytology samples of eight patients with congenital aniridia [age 34.5 ± 9.9 (17-51) years, 50% female] and eight healthy subjects [age 34.1 ± 11.9 (15-54) years, 50% female] were collected and analysed using mass spectrometry. Proteomic profiles were analysed in terms of molecular functions, biological processes, cellular components and pathway enrichment using the protein annotation of the evolutionary relationship (PANTHER) classification system. In total, 3323 proteins could be verified and there were 127 deregulated proteins (p < 0.01) in congenital aniridia. From the 127 deregulated proteins (DEPs), 82 altered biological processes, 63 deregulated cellular components, 27 significantly altered molecular functions and 31 enriched signalling pathways were identified. Pathological alteration of the biological processes and molecular functions of retinol binding and retinoic acid biosynthesis, as well as lipid metabolism and apoptosis related pathways could be demonstrated. Protein profile of conjunctival impression cytology samples of aniridia subjects identifies alterations of retinol binding, retinoic acid biosynthesis, lipid metabolism and apoptosis related pathways. Whether these changes are directly related to PAX6 haploinsufficiency, must be investigated in further studies. These new findings offer the possibility to identify potential new drug targets.

Dupilumab-associated ocular surface disease in atopic dermatitis patients: clinical characteristics, ophthalmic treatment response, and conjunctival goblet cell analysis.

Dupilumab-associated ocular surface disease (DAOSD) is frequently reported as side effect in atopic dermatitis (AD) patients. Therefore, the aim of this study was to investigate the frequency and severity of DAOSD, ophthalmic treatment response, and to learn more about the effect of dupilumab on conjunctival goblet cells (GC). This prospective study included dupilumab-treated AD patients between February 2020 and June 2022 from the University Medical Centre Utrecht. Patients were examined by an ophthalmologist and a dermatologist before start (baseline), and after 4 and 28 weeks of dupilumab treatment. Ophthalmological examination was assessed by the Utrecht Ophthalmic Inflammatory and Allergic disease (UTOPIA) score. DAOSD was defined as an increase in UTOPIA score of ≥3 points from baseline. To quantify conjunctival GCs and to investigate the percentage of Cytokeratin 19 (CK19)-CD45-Mucin 5AC (MUC5AC)+ cells, conjunctival impression cytology samples were analysed. Ocular surface disease (OSD) was present in 91.3% (n=63/69) patients at baseline. DAOSD was observed in 28.9% (n=20/69) patients, in whom GC numbers remained stable and the percentage of CK19-CD45-MUC5AC+ cells decreased at onset of DAOSD compared with baseline. After 28 weeks of dupilumab treatment, DAOSD was seen in 14.5% (n=10/69) patients. Of the 85.5% (n=59/69) patients without DAOSD or with controlled DAOSD at week 28, 40.7% (n=24/59) patients received anti-inflammatory ophthalmic drugs. OSD is common in moderate-to-severe AD patients before starting dupilumab. During treatment with dupilumab DAOSD severity improves with early ophthalmic treatment. The decrease in percentage of CK19-CD45-MUC5AC+ cells during dupilumab treatment suggests an impairment of the GC function due to dupilumab treatment.

Prolonged Computer Use by Office Workers Induces Ocular Symptoms Associated With Tear Film Alterations and Overexpression of Mucin 5 AC and Catalase.

The aim of the study was to evaluate office workers for symptoms of computer vision syndrome (CVS) and alterations in the tear film relate to the hours of daily computer use. Sixty-seven volunteers were divided into 2 groups: 2 to 6 and 7 to 12 hours of daily computer use. Computer vision syndrome symptoms, tear film stability by tear film break-up time test, and composition of mucin 5 AC, catalase, and IL-6 was assessed by relative gene expression of conjunctival impression cytology samples were examined. All participants exhibited moderate symptoms of CVS, whereas 90% showed reduced tear film stability. For the 7- to 12-hour (vs 2- to 6-hour) group, these effects were more pronounced and overexpression of mucin 5 AC and catalase was detected. Prolonged computer use induced an overexpression of mucin 5 AC and catalase and instability of the tear film, associated with ocular symptoms.

MiR-223 inhibits hyperosmolarity-induced inflammation through downregulating NLRP3 activation in human corneal epithelial cells and dry eye patients

We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1β expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3’ UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1β expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1β tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1β expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1β secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.

Local ocular surface findings in COVID-19 patients without ocular symptoms.

To investigate subjective ocular symptoms and objectively measure tear secretion in patients with a confirmed diagnosis of coronavirus disease-2019 (COVID-19). In this prospective cross-sectional study, 24 patients who had survived COVID-19 infection and 27 healthy controls were enrolled. Conjunctival impression cytology, the Schirmer test, tear-film break-up time, corneal staining scores were applied to all the participants. No significant difference was noted with regard to the gender and mean age between the two groups (p=0.484 and p=0.599, respectively). The conjunctival impression cytology analysis revealed that the density of the goblet cells was decreased, while the counts of lymphocytes and neutrophils were increased in the COVID-19 group patients when compared with ethe control group patients. When the Nelson classification was applied to the conjunctival impression cytology samples, 25% of the COVID-19 group patients and 14.8% of the control group patients exhibited changes consistent with ≥grade 2. The mean tear-film break-up time, Schirmer test, and corneal staining score results were determined to differ between the COVID-19 and control groups (p=0.02, p<0.001, and p=0.003, respectively). The present study revealed the pathological conjunctival alterations of patients with a confirmed diagnosis of COVID-19, indicating the possibility of the occurrence of pathological ocular surface alterations to even at the end of COVID-19 infection, without the occurrence of any significant clinical ocular manifestations.

Impact of Low Humidity on Damage-associated Molecular Patterns at the Ocular Surface during Dry Eye Disease.

Dry eye is one of the leading causes for individuals to seek eye care, whereas the pathogenesis is poorly understood. One mechanism in which dry eye inflammation may ensue is by the release of damage-associated molecular patterns (DAMPs) by damaged cells to stimulate the production of cytokines and matrix metalloproteinases. Examining DAMP levels on the ocular surface during dry eye disease (DED) will increase our understanding of their potential involvement in the pathogenesis of DED. This study aimed to quantitate DAMPs, high-mobility group box 1 (HMGB1), and heat shock proteins on the ocular surface of normal and dry eye subjects and to examine the impact of low-humidity environment (LHE) on DAMPs and inflammation in dry eye subjects. Basal tears (10 to 20 μL) and conjunctival impression cytology samples were analyzed for HMGB1, HSP-27, HSP-60, HSP-70, and HSP-90α by ELISA or Luminex assays in normal (n = 15) and DED (n = 15) subjects. In addition, a subset of DED subjects were exposed to LHE for 2 hours. The level of DAMPs in the tear film was evaluated by ELISA or Luminex assay. Interleukin 6, interleukin 8, or metalloproteinase (MMP) 9 mRNA were quantitated by real-time polymerase chain reaction from conjunctival impression cytology samples. Compared with age-matched normal subjects, HMGB1 was significantly elevated in the tear film of DED subjects (P = .03), whereas there was no significant difference in heat shock proteins. Conjunctival impression cytology samples revealed no significant difference in intracellular DAMP levels between both groups. After exposure to an LHE, there was an increase in corneal staining (P = .005), HSP-60 levels in the tear film (P = .01), and MMP-9 mRNA in the conjunctiva (P = .001). Dry eye subjects had higher levels of HMGB1 in their tear film. Exposure to an LHE worsened corneal staining, increased conjunctival MMP-9 mRNA expression, and increased tear film HSP-60 levels. Larger studies are needed to understand the involvement of DAMPs in stimulating dry eye inflammation.

Ocular Surface and Conjunctival Cytology Findings in Patients With Confirmed COVID-19.

To assess the effect of severe acute respiratory syndrome coronavirus-2 infection on the conjunctiva and tear film. Thirty-eight patients with confirmed COVID-19 and 31 healthy controls were included in this prospective and observational study. Individuals with COVID-19 formed the patient group, and healthy individuals formed the control group. Conjunctival impression cytology (CIC), TBUT, Schirmer II test, and ocular surface disease index were evaluated in all participants. No significant difference was observed regarding the mean age and gender between the groups (P=0.786 and P=0.122, respectively). The mean TBUT and Schirmer II test results did not differ between the two groups (P=0.496 and P=0.447, respectively). The CIC results revealed decreased density and cell size of goblet cells and moderate to high enlargement, squamous changes, and increased nucleocytoplasmic ratio in nongoblet epithelial cells in the COVID-19 group compared with the control group. Based on the Nelson classification in CIC samples, 60.6% of the COVID-19 group and 19.4% of the control group had changes consistent with grade 2 or above. The presence of neutrophils in CIC was significantly higher in the COVID-19 group (P<0.001), whereas the presence of lymphocyte was similar between the two groups (P=0.247). This study revealed the pathological conjunctival alterations in patients with COVID-19 and demonstrated that pathological ocular surface alterations may present even at the beginning of COVID-19 without clinically significant ocular manifestation.

Genistein-Calcitriol Mitigates Hyperosmotic Stress-Induced TonEBP, CFTR Dysfunction, VDR Degradation and Inflammation in Dry Eye Disease.

Dry eye disease (DED) signs and symptoms are causally associated with increased ocular surface (OS) inflammation. Modulation of key regulators of aberrant OS inflammation is of interest for clinical management. We investigated the status and the potential to harness key endogenous protective factors, such as cystic fibrosis transmembrane conductance regulator (CFTR) and vitamin D receptor (VDR) in hyperosmotic stress‐associated inflammation in patients with DED and in vitro. Conjunctival impression cytology samples from control subjects (n = 11) and patients with DED (n = 15) were used to determine the status of hyperosmotic stress (TonEBP/NFAT5), inflammation (IL‐6, IL‐8, IL‐17A/F, TNFα, MMP9, and MCP1), VDR, and intracellular chloride ion (GLRX5) by quantitative polymerase chain reaction and/or immunofluorescence. Human corneal epithelial cells (HCECs) were used to study the effect of CFTR activator (genistein) and vitamin D (calcitriol) in hyperosmotic stress (HOs)‐induced response in vitro. Western blotting was used to determine the expression of these proteins, along with p‐p38. Significantly, higher expression of inflammatory factors, TonEBP, GLRX5, and reduced VDR were observed in patients with DED and in HOs‐induced HCECs in vitro. Expression of TonEBP positively correlated with expression of inflammatory genes in DED. Increased TonEBP and GLRX5 provides confirmation of osmotic stress and chloride ion imbalance in OS epithelium in DED. These along with reduced VDR suggests dysregulated OS homeostasis in DED. Combination of genistein and calcitriol reduced HOs‐induced TonEBP, inflammatory gene expression, and p‐p38, and abated VDR degradation in HCECs. Henceforth, this combination should be further explored for its relevance in the management of DED.

Assessment of goblet cell size and density in relation to epithelial cell (multi)layering on conjunctival impression cytology samples.

To assess goblet cell size and numbers in relation to the extent of multilayering of conjunctival impression cytology (CIC) samples as a basis for reducing variability in image selection for goblet cell density (GCD) estimates. CIC was undertaken immediately postmortem off the superior bulbar conjunctiva of healthy young adult rabbits onto Millicell-CM Biopore filter units. After fixation with buffered glutaraldehyde and Giemsa staining, two × 200 images were selected from each sample representative of either slight multilayering or substantial multilayering, projected at × 1000, an overlay of the outlines of the goblet cells was made, and their dimensions and areas were measured. From measures of 4918 goblet cells, the average value (+/- SD) for the longest dimension was 17.7 ± 6.4μm and 14.6 ± 5.3μm for the shortest dimension. The GCD values ranged from 210 to 2069/mm2, with a mean of 1074 ± 601/mm2, but was lower for slightly multilayered images (at 537 ± 239 cells/mm) compared with multilayered regions (at 1612 ± 601 cells/mm2; p < 0.001). The measured areas ranged from 72 to 491μm2, with average values from any particular image ranging from 110 to 370μm2, which were inversely correlated with the estimated GCD (Spearman's rho = - 0.722, p < 0.05). Larger goblet cells but in fewer numbers were predictably found across the filter surface where there were fewer layers of cells and vice versa. This difference could be considered in selection of images for counts of goblet cells from CIC specimens.

Conjunctival HLA-DR Expression and Its Association With Symptoms and Signs in the DREAM Study.

PurposeEvaluation of dry eye disease (DED) relies on subjective symptoms and signs. We examined HLA-DR expression (HLA-DR%) in conjunctival cells, a minimally invasive biomarker with objective metrics, as an alternative method.MethodsDry Eye Assessment and Management (DREAM) study participants completed the Ocular Surface Disease Index questionnaire. Clinicians evaluated tear volume, tear breakup time, and corneal and conjunctival staining. Conjunctival impression cytology samples (n = 1049) were assessed for HLA-DR% in total cells (TCs), epithelial cells (ECs), and white blood cells (WBCs). Associations (categorized into <5%, 5%–15%, >15%–25%, and >25%) with symptoms and signs were evaluated.ResultsThe HLA-DR% varied markedly across samples. Over 40% had <5 HLA-DR% positive cells in TCs and ECs and under 23% in WBCs. Higher HLA-DR% was associated with higher conjunctival staining for ECs (mean score 2.77 for <5% and 3.28 for >25%, linear trend P = 0.009) and TCs (mean score 2.82 for <5% and 3.29 for >25%, linear trend P = 0.04) and in TCs was associated with higher corneal staining (mean score 3.59 for <5% and 4.46 for >25%, linear trend P = 0.03). HLA-DR% in WBCs did not correlated with signs (all P ≥ 0.58), and in TCs, ECs or WBCs were not associated with symptoms (P > 0.06).ConclusionsThe distribution of HLA-DR% in conjunctival cells reflects the heterogeneity of disease in DREAM participants. High percentages of samples with <5% positive cells indicate that HLA-DR% may not be a sensitive marker for DED in all patients.Translational RelevanceHigh HLA-DR% in ECs in association with high conjunctival staining may identify a subgroup of DED patients prone to epithelial disease and possibly need a different approach from current standards of treatment.

The Efficacy of 2% Topical Rebamipide on Conjunctival Squamous Metaplasia and Goblet Cell Density in Dry Eye Disease.

Purpose: To clarify the pharmacological effects of 2% rebamipide eye drops on mucosal membrane functions of the ocular surface epithelium, we investigated keratoconjunctival alterations at the cellular level in this study.Methods: Fifteen patients with definite dry eye disease were recruited from outpatient clinics of the Department of Ophthalmology, Ichikawa General Hospital. The patients received treatment with 2% rebamipide eye drops q.i.d for 12 weeks. Symptom score assessment, tear film breakup time, fluorescein and lissamine green ocular surface vital staining, grading of lid wiper epitheliopathy, Cochet–Bonnet corneal sensitivity, assessment of squamous metaplasia grades, and goblet cell density calculations from conjunctival impression cytology samples, as well as evaluation of nucleocytoplasmic ratios and corneal epithelial cells from in vivo confocal microscopy images before and 3 months after treatment were performed.Results: The mean symptom scores, tear film breakup time values, ocular surface fluorescein and lissamine green vital staining scores, and lid wiper scores showed a significant improvement after treatment (P < 0.01). The mean squamous metaplasia grade also showed a significant improvement (1.2 ± 0.1 → 0.3 ± 0.1) 3 months after treatment (P = 0.004). There were similar significant improvements in the mean corneal epithelial cell density (660.1 ± 62.6 → 1015.5 ± 43.5 cells/mm2) (P = 0.002) and nucleocytoplasmic ratios (0.1 ± 0.0 → 0.2 ± 0.0) (P = 0.0042) after treatment.Conclusions: Topical use of 2% rebamipide for 3 months was associated with improvements in ocular surface differentiation due to changes of mucosal functions at the cellular level. These alterations may explain objective and subjective improvements in dry eye disease.

Elevated Tear Human Neutrophil Peptides 1-3, Human Beta Defensin-2 Levels and Conjunctival Cathelicidin LL-37 Gene Expression in Ocular Rosacea

ABSTRACTPurpose: To investigate the role of innate immunity in ocular rosacea.Methods: Thirty-two patients with ocular rosacea patients (group-1) and 28 healthy volunteers (group-2) who served as controls were enrolled in the study. Tear function parameters were assessed, conjunctival impression cytology was performed and tear samples were collected. Human-neutrophil-peptides (HNP) 1–3 and human-beta-defensin-2 (hBD-2) levels were measured in tears by using ELISA tests. Cathelicidin leucin-leucin-37 (LL-37), hBD-2, human-beta-defensin-9 (hBD-9) gene expression levels were measured in the conjunctival impression cytology samples using real-time polymerase chain reaction.Results: Tear HNP1-3 (p = 0.024), hBD-2 (p < 0.001), conjunctival LL-37 gene expression rate (p = 0.014) and ocular surface disease index scores (p = 0.001) were higher and the tear break-up time was lower (p = 0.003) in group-1. No other differences were found between the groups.Conclusion: The results of this study suggest the role of abnormal innate immunity in the pathophysiology of ocular rosacea by revealing elevated antimicrobial peptide levels.

Conjunctival MUC5AC+ goblet cell index: relationship with corneal nerves and dry eye.

To evaluate the relative proportion of conjunctival MUC5AC+ and MUC5AC- goblet cells in a post-LASIK population and their association with dry eye indicators and corneal nerve morphology using a MUC5AC+ Goblet Cell Index. Twenty subjects who had undergone LASIK > 12months previously and 20 age-matched controls were recruited. Dry eye symptoms, tear breakup time, osmolarity, meniscus area and corneal nerve morphology were examined. Conjunctival impression cytology samples were collected from inferior-temporal bulbar conjunctiva using Millicell® inserts. Total goblet cell density was determined from positive cytokeratin-7 (CK7) immunolabelling; MUC5AC+ goblet cell density was determined from both CK7+- and MUC5AC+-immunolabelled cells. The ratio of MUC5AC+ to total density was defined as the "MUC5AC+ Goblet Cell Index". Differences in variables between groups and the associations between goblet cell variables and clinical assessments were examined. No significant differences in the total and MUC5AC+ goblet cell density and tear film parameters were found between groups, although greater ocular discomfort was reported in the post-LASIK group (P = 0.02). A higher MUC5AC+ Index was associated with worse/greater dry eye symptoms (ρ = 0.55, P = 0.01) and higher nerve tortuosity (ρ = 0.57, P = 0.01) in the post-LASIK group; lower nerve density and thickness was found in controls (ρ > -0.45, P < 0.05), but not associated with tear film parameters. The MUC5AC+ Goblet Cell Index provides an indicator of mucin secretion for assessing the goblet cell function in dry eye. In the post-LASIK participants, we found an increased MUC5AC+ Index associated with worse dry eye symptoms and adverse changes in corneal nerve morphology.

A Grid-Based Nucleus Counting Method for Estimates of the Density of Superficial Conjunctival Cells from Impression Cytology Samples Taken from Normal Healthy Human Eyes

ABSTRACTPurpose: To develop and validate an easily procured objective estimate of the cell density of the superficial epithelial cells of the normal bulbar conjunctiva using conjunctival impression cytology (CIC).Methods: From 20 healthy non-contact lens-wearing young adults, CIC was undertaken off the nasal aspect of the exposed bulbar conjunctiva, the filters stained with Giemsa, and images taken at 200× that only included an all-but-contiguous monolayer of epithelial cells. These images were projected at 1000× magnification and an overlay drawn of the location of the stained nuclei, from which two types of analysis were undertaken using either 100 micron circular or square regions of interest (ROI). From the former, the distances between all nuclei were measured and the number of nuclei/circle calculated to establish uniformity of cell nuclei distribution as a necessary prerequisite to using a grid-based counting method. From the latter, the number of nuclei in each square grid was manually counted and the grid-by-grid variability assessed.Results: The average inter-nucleus distance was 16.1+/3.5 microns, with predictable positioning to successive nuclei in the circular ROI to give an estimated numerical density of 1944 ± 237/sq mm. Counting of nuclei in grid squares yielded numbers between 12 and 42 to yield an estimated cell density of 2390 +/ ̶330 /sq mm, with repeat counts off grid square ROIs being predictably within +/–15%.Conclusions: These studies indicate that it should be fairly easy to use carefully selected CIC samples to make estimates of the density of the superficial conjunctival cells. Such a method should be useful and offer advantage over the subjective assignment of morphological grades to CIC samples which have been shown to be highly variable.

The analysis of human conjunctival epithelium proteome in ocular surface diseases using impression cytology and 2D-DIGE

Conjunctival impression cytology samples from patients with meibomian gland dysfunction (MGD), dry eye (DE), and healthy subjects (CT) were collected for determination of the degree of squamous metaplasia (SM) by PAS-hematoxylin staining and for comparative proteomic analyses by 2D-DIGE. The protein spots with discriminant expression were identified by MALDI-TOF/TOF mass spectrometry. Three independent statistical studies were conducted: i). Analysis of differential protein expression between study groups: We observed increased expression of proteins S100A4, S100A8, retinal dehydrogenase-1, peroxiredoxin-1, annexin-A1, annexin-A2, α-enolase, and glutathione S-transferase-P in DE, whereas the highest expression of peroxiredoxin-6, actin cytoplasmic-1, peroxiredoxin-2, and heat shock protein HSP-90-α was observed in MGD; ii). Correlation between changes in the proteome profile and the grade of SM: The expression of 5 different cytokeratins (KRT1, KRT4, KRT8, KRT10, and KRT13) correlated with the degree of SM; iii). Proteome profile differences between pathological and CT groups: An overall proteome analysis revealed upregulation of 9 proteins in the pathological groups (Annexin-A1, α-enolase, Annexin-A2, S100A8, cytokeratin-1, Peroxiredoxin-2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin-3 and Lipocalin-1). In conclusion, a sensitive proteomic approach to study conjunctival tissue collected from minimally invasive impression cytology was implemented. Differential proteomics analyses showed that in comparison with the MGD, the DE patients presented higher overexpression of proteins related to antimicrobial defense, tissue-damage response, and regulation of body fluid secretions. Changes in MGD proteome were associated with oxidative stress and anti-apoptotic processes. We found a correlation between the grade of SM and expression of proteins associated with cytoskeleton and keratinization. The studied pathological groups shared elements related to the defense and inflammatory responses. Dot blot assays of proteins ANXA1, S100A8, and S100A4 validated the proteomic results obtained from 2D-DIGE experiments and confirmed the correlation between the expression of these proteins and the clinical parameters.

Evaluation of the Tear Function Tests and the Ocular Surface in First-Time Users of Silicone Hydrogel Contact Lenses

ABSTRACTPurpose: To evaluate the effects of three different silicone hydrogel contact lenses (SHCL), (balafilcon A, senofilcon A, and comfilcon A) on tear function tests, corneal thickness, and ocular surface cytology in first time contact lens users.Materials and methods: In this prospective study, 120 eyes of 60 subjects were evaluated. Balafilcon A users were designated as group 1, senofilcon A users as group 2, and comfilcon A users as group 3. In all cases, before and after 6 months of contact lens wear, ocular surface disease index score (OSDI), tear breakup time (TBUT), Schirmer 1 test, central corneal thickness (CCT), central corneal epithelium thickness (CCET), and conjunctival impression cytology samples were evaluated.Results: In group 1, 40 eyes of the 20 patients, in group 2, 40 eyes of the 20 patients, and in group 3, 40 eyes of the 20 patients were evaluated. The mean OSDI scores did not differ between the three groups after contact lens wear (p > 0.05). In group 1 and group 2, significant decrease was found in the mean TBUT 6 months after contact lens wear (p = 0.04, p < 0.001, respectively). In group 3, after 6 months of contact lens wear, the mean Schirmer 1 tear test was decreased significantly (p = 0.021). In all 3 groups, no significant change was observed in the mean CCT and CCET after contact lens wear (p > 0.05). After 6 months, the morphological changes in temporal and superior conjunctival epithelial cells were found to be significant in all groups (p < 0.001).Conclusion: Six months after SHCL wear, marked morphological changes occurred in the conjunctival epithelium. Tear function tests were also affected, while corneal thickness did not show any significant difference.

Clinicopathological Correlation in Dry Eye Disease

Introduction: To establish the strength of the association between routine tear function tests and conjunctival impression cytology (CIC) and to determine whether they simulate the morphological and cytological changes that occur on the ocular surface in dry eye. Materials and Methods: Fifty six patients (30 cases and 26 controls) were included in the study. The tear film profile included tear film break up time (TBUT), Schirmer’s-1, Rose Bengal scores (RBS), and impression cytology. CIC samples were obtained from the infero-temporal quadrant of bulbar conjunctiva and stained with Papanicolaou stain (PAP) and Periodic Acid Schiff (PAS) stains. Results: In our study, among clinical tests RBS (86.7%) had maximum sensitivity and highest percentage of specificity of TBUT (88.5%). Predictive values of Schirmer’s test, RBS, GCD, TBUT and CIC was 77.8%, 83.9%, 87.9%, 88.9% and 93.8% respectively in our study group. Cytological findings correlated well with histological findings. Conclusion: Impression cytology was found to be better when compared to clinical tests. These etiopathological findings will help to decide proper line of management.

Dry eye modulates the expression of toll-like receptors on the ocular surface

We aimed to determine if toll-like receptor (TLR) expression is modulated in response to dry eye-associated conditions and in dry eye syndrome (DES). Primary human corneal epithelial cells (HCEC), an SV40 HCEC cell line or a normal human conjunctival epithelial cell line (IOBA-NHC) were cultured under hyperosmolar stress (HOS) (400–500 mOsm/kg) or with DES associated cytokines (IL-1α/β, TNFα or TGFβ) at concentrations ranging from 1 to 1000 ng/ml for up to 24 h. Epithelial cells were harvested from a human cornea organ culture model following 24 h of desiccation. Conjunctival impression cytology samples were harvested from subjects with DES and age and gender-matched normal subjects. TLR4, TLR5 or TLR9 mRNA or protein was examined by quantitative RT-PCR, western blotting or flow cytometry. TLR functionality was evaluated in terms of addition of TLR agonists and quantitation of secreted inflammatory cytokines by the use of ELISA and Luminex assays. In SV40 HCEC, HOS significantly increased TLR4 by 8.18 fold, decreased TLR9 by 0.58 fold, but had no effect on TLR5 mRNA expression. TLR4 and TLR9 protein were decreased by 67.7% and 72% respectively. TLR4 mRNA was also significantly up-regulated by up to 9.70 and 3.36 fold in primary HCEC and IOBA-NHC respectively. DES associated cytokines had no effect on TLR4, TLR5 and TLR9 expression. In response to desiccation, TLR4 and TLR5 mRNA were significantly up-regulated by 4.81 and 2.51 fold respectively, while TLR9 mRNA was down-regulated by 0.86 fold in HCEC. A similar trend for TLR4 and TLR9 protein was observed. TLR9 mRNA was significantly down-regulated by almost 59.5% in DES subjects. In conclusion, changes in TLR expression occur in dry eye and could have an important role in ocular surface susceptibility to inflammation and infection.

Polymorphism in THBS1 Gene Is Associated with Post-Refractive Surgery Chronic Ocular Surface Inflammation

To determine the association of single nucleotide polymorphisms (SNPs) of the thrombospondin 1 (THBS1) gene with development of chronic ocular surface inflammation (keratoconjunctivitis) after refractive surgery. Retrospective cohort study. Active duty U.S. Army soldiers (n = 143) who opted for refractive surgery. Conjunctival impression cytology samples collected from participants before the surgery were used to harvest DNA for genotyping 5 THBS1 SNPs (rs1478604, rs2228262, rs2292305, rs2228262, and rs3743125) using the Sequenom iPLEX Gold platform (Sequenom, San Diego, CA). Samples collected after surgery were used to harvest RNA for gene expression analysis by real-time polymerase chain reaction (PCR). Participants were followed for 1 year after surgery to monitor the status of keratoconjunctivitis. Genetic basis of the development of chronic keratoconjunctivitis after refractive surgery. Carriers of minor alleles of 3 SNPs each were found to be more susceptible to developing chronic keratoconjunctivitis (rs1478604: odds ratio [OR], 2.5; 95% confidence interval [CI], 1.41-4.47; P = 2.5 × 10(-3); rs2228262 and rs2292305: OR, 1.9; 95% CI, 1.05-3.51; P = 4.8 × 10(-2)). Carriers of the rs1478604 minor allele expressed significantly reduced levels of thrombospondin 1 (TSP1) (P = 0.042) and increased levels of an inflammatory cytokine associated with keratoconjunctivitis, interleukin-1β (P = 0.025), in their ocular surface epithelial cells compared with homozygous major allele controls. Genetic variation in the THBS1 gene that results in decreased expression of the encoded glycoprotein TSP1 in ocular surface epithelial cells significantly increases the susceptibility to develop chronic ocular surface inflammation after refractive surgery. Further investigation of THBS1 SNPs in a larger sample size is warranted.