Alteration of Gene Expression in Pathological Keratinization of the Ocular Surface.

Purpose: To investigate the molecular mechanism of pathological keratinization in the chronic phase of ocular surface (OS) diseases. Methods: In this study, a comprehensive gene expression analysis was performed using oligonucleotide microarrays on OS epithelial cells obtained from three patients with pathological keratinization (Stevens-Johnson syndrome [n = 1 patient], ocular cicatricial pemphigoid [n = 1 patient], and anterior staphyloma [n = 1 patient]). The controls were three patients with conjunctivochalasis. The expression in some transcripts was confirmed using quantitative real-time PCR. Results: Compared to the controls, 3118 genes were significantly upregulated by a factor of 2 or more than one-half in the pathological keratinized epithelial cells (analysis of variance P < 0.05). Genes involved in keratinization, lipid metabolism, and oxidoreductase were upregulated, while genes involved in cellular response, as well as known transcription factors (TFs), were downregulated. Those genes were further analyzed with respect to TFs and retinoic acid (RA) through gene ontology analysis and known reports. The expression of TFs MYBL2, FOXM1, and SREBF2, was upregulated, and the TF ELF3 was significantly downregulated. The expression of AKR1B15, RDH12, and CRABP2 (i.e., genes related to RA, which is known to suppress keratinization) was increased more than twentyfold, whereas the expression of genes RARB and RARRES3 was decreased by 1/50. CRABP2, RARB, and RARRES3 expression changes were also confirmed by qRT-PCR. Conclusions: In pathological keratinized ocular surfaces, common transcript changes, including abnormalities in vitamin A metabolism, are involved in the mechanism of pathological keratinization.

Conjunctival transcriptomics in ocular mucous membrane pemphigoid.

PURPOSE: Ocular Mucous Membrane Pemphigoid (OcMMP) is an orphan disease characterized by chronic autoimmune-driven conjunctival inflammation leading to progressive scarring, debilitating symptoms, and blinding sequelae. This feasibility study aims to demonstrate conjunctival genetic transcriptomic analyses as a putative tool for interrogation of pathogenic signaling pathways in OcMMP. METHODS: Conjunctival RNA profiling using the NanoString nCounter Human Fibrosis panel was undertaken on RNA extracted from conjunctival swabs obtained from 6 MMP patients (8 eyes; 4 M/2F; median age 78 [range 64-84] years); and 8 age-matched control participants (15 eyes; 3 M/5F; median age 69.5 [range 69-88] years). Data from 770 genes were analyzed with ROSALIND HyperScale architecture and stratified according to the level of clinically visible bulbar conjunctival inflammation. Normalization, fold-changes (≥+1.5-fold or ≤ -1.5-fold) and p-values adjustment (<0.05) using the Benjamini-Hochberg method were calculated. RESULTS: 93 differentially expressed genes (DEGs) were observed between OcMMP versus controls of which 48 were upregulated, and 45 downregulated. The top 4 upregulated DEGs represented fibrosis (COL3A1, COL1A1, FN1 and THBS1) while the key under-expressed genes (SCIN, HMGS2, XCL1/2) were indicative of ocular surface failure (goblet cell loss, keratinization, vulnerability to secondary infections). Forty-four pathways had a global significance score ≥2, the most significant being those related to extracellular matrix (ECM) remodeling, synthesis, and degradation. These pathways were accentuated in eyes with visible inflammation. CONCLUSIONS: NanoString methodology acquired via a simple conjunctival swab identifies profibrotic genes in OcMMP group and differentiates inflamed eyes. Longitudinal sampling and following investigative intervention will further mechanistic insight and development of novel biomarkers to monitor disease progression.

Qi Jing Mingmu decoction inhibits the p38 signaling pathway in conjunctivochalasis fibroblasts by down-regulation of Th17 cell differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: Qi Jing Mingmu (QJMM) decoction is a traditional Chinese medicine that has been widely used for the clinical treatment of conjunctivochalasis (CCH). It is an effective treatment to relieve ocular symptoms including improving tear film and promoting tear secretion. However, its effects and molecular mechanisms need to be elucidated. AIM OF THE STUDY: To determine whether QJMM decoction affected T helper 17 (Th17) cell differentiation of CCH patients. MATERIALS AND METHODS: Blood samples and conjunctival tissues were collected from CCH patients and normal controls. The fibroblasts were separately induced, and CD4+ T cells were incubated with increasing concentrations of QJMM decoction and co-cultured with CCH fibroblasts. Th17 cell numbers were then analyzed using flow cytometry. Serum levels of interleukin 17 (IL-17) and IL-22 were detected using enzyme-linked immunosorbent assays. The expressions of signal proteins and genes were detected using western blotting and quantitative real-time PCR. RESULTS: Compared with normal controls, Th17 cell numbers and serum levels of IL-17 and IL-22 were elevated in patients with CCH. QJMM decoction down-regulated the expressions of IL-17, IL-22, and STAT3 of CD4+T cells from CCH patients, suggesting that QJMM decoction impeded Th17 cell differentiation. QJMM decoction-treated CD4+ T cells inhibited the expression of p38 in CCH fibroblasts. CONCLUSION: QJMM decoction inhibited Th17 cell differentiation of CD4+T cells from CCH patients, and QJMM decoction-treated CD4+T cells down-regulated the p38 signal pathway in CCH fibroblasts. Our study showed that Th17 cells may be good candidates for clinical treatment of CCH.

The conjunctival extracellular matrix, related disorders and development of substrates for conjunctival restoration.

The conjunctiva can be damaged by numerous diseases with scarring, loss of tissue and dysfunction. Depending on extent of damage, restoration of function may require a conjunctival graft. A wide variety of biological and synthetic substrates have been tested in the search for optimal conditions for ex vivo culture of conjunctival epithelial cells as a route toward tissue grafts. Each substrate has specific advantages but also disadvantages related to their unique physical and biological characteristics, and identification and development of an improved substrate remains a priority. To achieve the goal of mimicking and restoring a biological material, requires information from the material. Specifically, extracellular matrix (ECM) derived from conjunctival tissue. Knowledge of the composition and structure of native ECM and identifying contributions of individual components to its function would enable using or mimicking those components to develop improved biological substrates. ECM is comprised of two components: basement membrane secreted predominantly by epithelial cells containing laminins and type IV collagens, which directly support epithelial and goblet cell adhesion differentiation and growth and, interstitial matrix secreted by fibroblasts in lamina propria, which provides mechanical and structural support. This review presents current knowledge on anatomy, composition of conjunctival ECM and related conjunctival disorders. Requirements of potential substrates for conjunctival tissue engineering and transplantation are discussed. Biological and synthetic substrates and their components are described in an accompanying review.

Mechanism of Akt regulation of the expression of collagens and MMPs in conjunctivochalasis.

Akt is a central node of many signaling pathways, which plays important roles in cell survival, proliferation, migration, metabolism and collagen synthesis. Conjunctivochalasis (CCH) is one of the most common age-related ocular superficial diseases related to abnormalities in conjunctival extracellular matrix. Here, we studied the role of Akt regulating collagens and MMPs in the pathogenesis of CCH. Primary conjunctival fibroblasts were obtained from CCH patients (n = 13) and age-matched normal controls (n = 10). The levels of Akt, collagen type I, collagen type III, MMP1, and MMP3 were determined by Western blot, qRT-PCR, immunohistochemistry, and immunofluorescence staining. Normal control conjunctival fibroblasts were treated with Akt inhibitor A6730, and CCH fibroblasts were transfected with Akt overexpression vector. The expression of Akt in CCH was significantly lower than that in normal control of conjunctival tissues and cultured fibroblasts. Blocking Akt signaling with Akt inhibitor could inhibit the expression of collagen type I and collagen type III and upregulate the expression of MMP1 and MMP3. Meanwhile, compared with CCH fibroblasts transfected with control mimics, the protein and mRNA expression of collagen type I and collagen type III were increased significantly in Akt overexpression group, while the results of MMP1 and MMP3 in transfected fibroblasts were opposite. Taken together, Akt upregulated the expression of collagen type I and collagen type III and downregulated the expression of MMP1 and MMP3. Akt signaling pathway could provide a direct negative contribution to CCH and might be an attractive target for CCH therapy.

Local Renin-Angiotensin System Activation and Myofibroblast Formation in Graft Versus Host Disease-Associated Conjunctival Fibrosis.

Purpose: The present study was designed to investigate the role of myofibroblast transdifferentiation and the conjunctival renin-angiotensin system (RAS) in the pathogenesis of graft-versus-host disease (GVHD)-associated conjunctival fibrosis. Methods: A mouse model of major histocompatibility-matched allogeneic transplantation was used to induce GVHD, with male B10.D2 mice as donors and female BALB/c mice as recipients. Male BALB/c to female BALB/c syngeneic transplantation was used as control. Y chromosome staining in the spleen cells obtained from female recipient mice was used to confirm engraftment. The phenol red thread test and fluorescein staining were used to quantify tears and corneal keratopathy. Eyes were harvested at 4 and 8 weeks after the transplant for alpha-smooth muscle actin (α-SMA), angiotensinogen, and angiotensin-converting enzyme (ACE) immunostaining. Conjunctiva was harvested for gene expression quantification of α-SMA, angiotensinogen, and ACE. Results: More than 80% of the spleen cells in the recipient mice were chromosome Y positive, thus conforming successful engraftment. A significant decrease in tear secretion and a marked increase in corneal keratopathy score after allogeneic transplantation indicated the onset of ocular GVHD in these mice. A significant increase in α-SMA gene expression and the presence of a large number of α-SMA-positive cells was noted in the bulbar orbital conjunctiva of mice after allogeneic transplantation. Allogenic transplantation also caused a significant increase in the gene expression and protein expression of angiotensinogen and ACE in the subconjunctival eyelid area. Conclusions: Results of the present study demonstrate that GVHD-associated conjunctival fibrosis is accompanied by myofibroblast formation and activation of the local conjunctival RAS.

hADSCs derived extracellular vesicles inhibit NLRP3inflammasome activation and dry eye.

The present study was set out to address the therapeutic efficacy of human adipose tissue stem cells derived extracellular vesicles (hADSC-Evs) in a mouse model of dry eye disease and to investigate the underlying mechanisms involved. hADSC-Evs eye drops were topically administered to mice that subjected to desiccating stress (DS). Clinical parameters of ocular surface damage were assessed with fluorescein staining, tear production and PAS staining. For in vitro studies, cell viability assay and TUNEL staining were performed in human corneal epithelial cells (HCECs) treated with hADSC-Evs under hyperosmotic media. In addition, immunofluorescent staining, Real-time PCR (qRT-PCR) and Western blots were used to evaluated NLRP3, ASC, caspase-1, and IL-1ß expression levels. Compared with vehicle control mice, topical hADSC-Evs treated mice showed decreased corneal epithelial defects, increased tear production, decreased goblet cell loss, as well as reduced inflammatory cytokines production. In vitro, hADSC-Evs could protect HCECs against hyperosmotic stress-induced cell apoptosis. Mechanistically, hADSC-Evs treatment suppressed the DS induced rises in NLRP3 inflammasome formation, caspase-1 activation and IL-1ß maturation. In conclusion, hADSC-Evs eye drops effectively suppress NLRP3 inflammatory response and alleviate ocular surface damage in dry eye disease.

Paediatric orbital conjunctival epithelial cyst with positive asialotransferrin.

Conjunctival orbital cysts are rare; they are typically either conjunctival dermoid or conjunctival epithelial cysts - congenital or acquired (inclusion). We describe the case of a 15-month-old girl presenting with strabismus and proptosis who had a retrobulbar intraconal cystic lesion displacing the optic nerve, with an adjacent middle cranial fossa anomaly. Aspiration of the orbital cyst tested positive for asialotransferrin, raising the suspicion of a direct communication with cerebrospinal fluid (CSF). Subsequent fine cut CT scanning disproved any connection with the intracranial space, and the cyst was excised complete and intact. Histopathology showed a conjunctival epithelial cyst. To our knowledge, this is the first case report in the literature of an asialotransferrin positive pediatric orbital conjunctival epithelial cyst. It is of clinical relevance as it explores the possibility of either a false positive asialotransferrin or potentially a prior developmental communication with the subarachnoid space. These two diagnostic possibilities are discussed.

Chemokine Receptor Expression Pattern Correlates to Progression of Conjunctival Melanocytic Lesions.

Purpose: Chemokines play a role in the progression and metastatic spread of both cutaneous and uveal melanomas. The aim of this study was to examine the prognostic value of expression of chemokine receptors CCR7, CXCR4, and CCR10 in conjunctival melanocytic lesions. Methods: In total, 44 conjunctival nevi, 21 cases of primary acquired melanosis (PAM) with atypia and 35 conjunctival melanomas, were included. After immunohistochemical staining for CCR7, CXCR4, and CCR10 the immunoreactive score (IRS) was determined. The findings were correlated for association with melanoma and development of metastasis. For mechanistic evaluation, we used a mouse melanoma metastasis model using two human conjunctival melanoma cell lines, CM2005.1 and CRMM1. Results: All tested chemokines showed a significantly higher expression in conjunctival melanoma than conjunctival nevi. There was a statistically significant difference between the IRS in nevi and PAM with atypia for nuclear IRS in CCR10 (P = 0.03) and both nuclear and cytoplasmic IRS in CXCR4 (P < 0.01 and P = 0.03, respectively); this was also true evaluating the groups PAM with atypia and melanoma all together (P < 0.01). Furthermore, a trend for lower IRS was seen in cases of melanoma without metastasis, with a suggestive pattern of a higher IRS in cases that did develop metastases, supported for CXCR4 using the mouse melanoma metastasis model. Conclusions: Expression of specific chemokines changes during the progression and metastatic spread of conjunctival melanocytic lesions. Differential chemokine profiles may hold prognostic value for patients with conjunctival melanomas and might be considered as a therapeutic target.

Transforming Growth Factor-ß1-induced Human Subconjunctival Fibrosis is Mediated by MicroRNA 143/145 Expression.

Purpose: To investigate the roles and pathways of microRNAs 143 and 145 in transforming growth factor (TGF)-ß1-induced human subconjunctival fibrosis. Methods: Human tenon's capsule fibroblasts (HTFs) were obtained from a healthy eye. After treating cultured HTFs with TGF-ß1, the expression of microRNAs 143 and 145 was evaluated using polymerase chain reaction. To identify the pathways of TGF-ß1-induced microRNA 143/145 expression, HTFs were treated with specific inhibitors of p38MAPK, PI3K/Akt, JNK, ERK, and with siRNAs for SMAD2 and SMAD4. Mutagenesis studies were performed to evaluate the role of the CArG box and SMAD-binding element (SBE). To investigate the role of microRNA 143/145 in TGF-ß1-induced myofibroblast transdifferentiation, microRNA 143/145 mimics and microRNA 143/145 inhibitors were applied to the HTFs. Results: Array analysis revealed that TGF-ß1 induced the expression of microRNA 143/145 in a dose- and time-dependent manner. When inhibitors and siRNAs for p38MAPK, PI3K/Akt, ERK, and JNK were applied, the TGF-ß1-induced expression of microRNA 143/145 was inhibited; however, SMAD2 and SMAD4 inhibition did not affect the TGF-ß1-induced expression of these microRNAs. In the mutagenesis studies, both the CArG box and SBE were associated with TGF-ß1-induced expression of microRNA 143/145. Mimics of microRNA 143/145 induced increased myofibroblast formation, whereas their inhibitors had the opposite effect. Conclusions: TGF-ß1-induced human subconjunctival fibrosis was mediated by the expression of microRNA 143/145, mainly via SMAD-independent pathways. Inhibition of TGF-ß1-induced microRNA 143/145 expression in HTFs might represent a novel strategy to prevent subconjunctival fibrosis.

The Effect of Rebamipide Ophthalmic Solution on Cytokine and Mucin Secretion in Culture of Conjunctival Epithelial Cells From the Cu, Zn-Superoxide Dismutase-1 (SOD-1) Knock-Down Mouse.

OBJECTIVES: To evaluate the in vitro effects of 1-mM rebamipide ophthalmic solution on the expression of inflammatory cytokines and MUC5AC in Cu, Zn-superoxide dismutase-1 (SOD-1) knock-down conjunctival epithelium. METHODS: Conjunctival epithelium from C57BL/6 wild-type mice was cultured and treated with rebamipide ophthalmic solution. Using cytometric bead array, we examined the levels of interleukin-(IL)-6, IL-10, IL-17, monocyte chemoattractant protein-1, interferon-γ (INF-γ), tumor necrosis factor, and IL-12p70 in the culture supernatants. The culture supernatants were obtained from the culture medium of nontreated or SOD-1 knock-down conjunctival epithelium using small interfering RNA (siRNA). In addition, ELISA was performed to ascertain the MUC5AC concentration in the culture medium. RESULTS: After rebamipide ophthalmic solution was applied, IL-6 concentration in the supernatants of conjunctival epithelial cells treated with and without siRNA showed a significant timewise decrease from 0 to 24 hr (963±42 to 0.07±0.05 pg/mL and 932±168 to 2.2±0.05 pg/mL, respectively) (P<0.001). Compared with baseline values, MUC5AC concentrations significantly increased 24 hr after rebamipide application to the conjunctival cultures-both with and without SOD-1 siRNA treatment (P<0.05 in both cases). CONCLUSIONS: Rebamipide seems to increase MUC5AC levels and suppress inflammation by decreasing IL-6 levels in mouse conjunctival epithelial cell cultures. SOD-1 siRNA-treated mouse conjunctival epithelial cell culture is a practical method for investigating changes in mucosa-associated mucins and proinflammatory cytokines in response to therapeutic interventions.

Ocular Impression-Taking-Which Material Is Best?

OBJECTIVES: To assess the efficacy and effect on clinical signs of a polyvinylsiloxane (Tresident; Shütz Dental Group GmbH, Germany) compared with an irreversible hydrocolloid (Orthoprint; Zhermack SpA, Badia Polesine, Italy) for ocular impression-taking. METHODS: Twenty subjects were recruited (13 female and 7 male), with mean age 31.1±4.6 years (SD) (range 25.8-39.7). Subjects attended for 2 sessions, each of 1-hr duration, on 2 separate days. Each session was scheduled at the same time on each day. At each visit, the subject underwent an ocular impression procedure, using either Tresident or Orthoprint, in random order and to one eye only. Investigator 2 was blind to this assignment. Two experienced practitioners conducted the study, investigator 1 performed the ocular impression procedures and investigator 2 observed and assessed the clinical signs: logMAR visual acuity, ocular surface staining, tear break-up time (TBUT), and ocular hyperemia. RESULTS: Visual acuity was unaffected by either material; TBUT was marginally disrupted by both materials, but was not clinically significant according to published criteria; ocular redness increased with both materials; and corneal staining was significantly greater after Orthoprint impression. Less redness and clinically insignificant staining after impression-taking, with fewer clinical complications, was found after use of Tresident. CONCLUSIONS: Tresident offers a quicker, more effective, and clinically viable method of obtaining ocular impression topography compared with the traditional Orthoprint, and Orthoprint causes significantly more superficial punctuate staining of the corneal epithelium than Tresident.

Epithelial dysplasia in pterygium postoperative granuloma.

Pterygium postoperative granuloma (PPG) is one of the common complications of pterygium surgery. In order to provide the structural features of PPG, and to further explore its pathogenetic mechanism, we analyzed clinical and pathological characteristics of 12 PPG cases. New blood vessels were observed under a slit lamp in PPG and peripheral conjunctival tissues. In vivo confocal imaging showed that there was extensive neovascularization in the stroma, accompanied by infiltration of dendritic cells and inflammatory cells. Dense fibrous structures were observed in some PPG tissues. H&E staining results confirmed neovascularization and inflammatory cells in PPG tissues. In addition, H&E staining exhibited epithelioid tissue covering some PPG tissues. The immunofluorescence results demonstrated that the PPG epithelium was negative for K19, K10 and Muc5AC. Compared with the normal conjunctiva and pterygium, the expression of collagen IV in PPG basement membrane decreased, the expression of pan-cytokeratin (PCK), claudin 4 and E-cadherin in PPG epithelium was significantly lower, while the expression of vimentin, α-SMA and Snail was significantly increased. Therefore, our results suggest that the expression of epithelial keratin markers and goblet cell specific mucin marker is downregulated in the PPG tissues, and it likely is associated with the occurrence of EMT in granulomatous tissues.

Context-Dependent Regulation of Conjunctival Goblet Cell Function by Allergic Mediators.

In the eye, goblet cells responsible for secreting mucins are found in the conjunctiva. When mucin production is not tightly regulated several ocular surface disorders may occur. In this study, the effect of the T helper (Th) 2-type cytokines IL4, IL5, and IL13 on conjunctival goblet cell function was explored. Goblet cells from rat conjunctiva were cultured and characterized. The presence of cytokine receptors was confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Changes in intracellular [Ca2+], high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with Th2 cytokines with or without the allergic mediator histamine. We found that IL4 and IL13 enhance cell proliferation and, along with histamine, stimulate goblet cell secretion. We conclude that the high levels of IL4, IL5, and IL13 that characterize allergic conjunctivitis could be the reason for higher numbers of goblet cells and mucin overproduction found in this condition.

Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition.

BACKGROUND: To develop targeted antifibrotic therapy for glaucoma filtration surgery; this study determines the effectiveness of small interfering RNA (siRNA) to reduce in vivo secreted protein acidic and rich in cysteine (SPARC) expression using the mouse model of conjunctival scarring. METHODS: Experimental surgery was performed as described for the mouse model of conjunctival scarring. Scrambled (siScram) or Sparc (siSparc) siRNAs, loaded on layer-by-layer (LbL) nanoparticles, were injected into the conjunctiva immediately after surgery. Expression of Sparc, Col1a1, Fn1 and Mmp14 was measured by real-time PCR and immunoblotting on days 7 and 14 postsurgery. Live imaging of the operated eyes was performed using slit lamp, anterior segment-optical coherence tomography and confocal microscopy. Tissue pathology was evaluated by histochemical and immunofluorescent analyses of operated conjunctival cryosections. Tissue apoptosis was quantitated by annexin V assay. RESULTS : siSparc, delivered via expanded LbL nanoparticles, significantly inhibited Sparc transcription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues. Sparc suppression on day 7 was associated with a significant reduction of Col1a1 (2.52-fold), Fn1 (2.89-fold) and Mmp14 (2.23-fold) mRNAs. At the protein level, both SPARC and collagen 1A1 (COL1A1) were significantly reduced at both time points with siSparc treatment. Nanoparticles were visualised within cell-like structures by confocal microscopy, while overt tissue response or apoptosis was not observed. CONCLUSIONS : SPARC targeted therapy effectively reduced both SPARC and collagen production in the operated mouse conjunctiva. This proof-of-concept study suggests that targeted treatment of fibrosis in glaucoma surgery is safe and feasible, with the potential to extend to a range of potential genes associated with fibrosis.

Ocular Adnexal Amyloidosis: A Mass Spectrometric Analysis.

PURPOSE: Ocular adnexal amyloidosis (OAA) may represent localized manifestation of an underlying systemic process. Accurate identification of the amyloid fibrils can guide the systemic evaluation and treatment. The aim of this study was to characterize subtypes of OAA using immunohistochemistry and mass spectrometric analysis and to correlate with ocular involvement and systemic association. DESIGN: Retrospective case series. METHODS: Review of patients with OAA subtyped by immunohistochemistry and mass spectrometric analysis at the Cleveland Clinic from June 1995 to June 2017. RESULTS: While immunohistochemistry identified AL amyloid protein in 67% (4/6) of specimens tested, mass spectrometry identified AL amyloid protein in all specimens (10/10). AL lambda was identified in 5 (50%) samples, kappa in 3 (30%), and both kappa and lambda light chains in 2 (20%). The 5 cases of conjunctival amyloidosis were either AL lambda only (3 cases) or both lambda and kappa (2 cases). There were 3 cases that had associated systemic involvement. Two of these had eyelid skin involvement and AL kappa amyloidosis and the other patient had uveal involvement and AL lambda amyloidosis. CONCLUSIONS: Primary amyloidosis-AL is the most common form diagnosed by mass spectrometric analysis in patients with OAA. Immunohistochemistry is ineffective in the characterization of the amyloid deposits in a significant number of cases. Evaluation to exclude systemic involvement or associated underlying lymphoproliferative disorder is warranted.

Topical fluorometholone treatment and desiccating stress change inflammatory protein expression in tears.

PURPOSE: It was hypothesized that tear protein biomarkers could predict the effects of topical steroid treatment and desiccating stress in patients with dry eye disease (DED). To test this concept, a randomized, double-masked, controlled clinical trial with 41 patients was conducted. METHODS: The patients were treated topically with either 0.1% fluorometholone (FML) or polyvinyl alcohol (PA). Tear samples were collected using 1 µl glass capillaries at recruitment into the study and after a 3-week treatment period, both before and after 2 h exposure to desiccating stress, in a controlled environment chamber. Relative quantification of tear proteins was conducted by NanoLC-MSTOF using sequential window acquisition of all theoretical mass spectra (SWATH). Ocular surface integrity (corneal and conjunctival staining and conjunctival hyperemia) was selected as the key DED-related sign and analyzed with proteomic data. Analysis of covariance (ANCOVA) and linear models were used to analyze the data with R. RESULTS: 758 proteins were identified and relatively quantified from each tear sample. Analysis revealed 9 differentially expressed proteins between FML and PA treatments after 3 weeks and 7 after desiccating stress (P < 0.05). We also identified several differentially expressed proteins at the initial collection, which could be used to predict changes of conjunctival and corneal staining and conjunctival hyperemia after FML treatment and after desiccating stress. These proteins include complement C3 (C3) and calmodulin like 5 (CALML5), which could also differentiate the severity of DED at baseline. CONCLUSIONS: The identified proteins could be further used as biomarkers to identify patients most benefiting from FML treatment.

Factors that influence tear meniscus area and conjunctivochalasis: The Singapore Indian eye study.

PURPOSE: Assessment of tear film and conjunctiva is critical to define presence and severity of ocular surface disease. We aimed to characterize tear meniscus area (TMA) and conjunctivochalasis by anterior segment optical coherence tomography (ASOCT) in population-based patients and identify potential factors associated with low TMA and severe conjunctivochalasis. METHODS: Study subjects were enrolled from The Singapore Indian Eye Study, a population-based study of Asian Indian in Singapore. Imaging with ASOCT was performed on three ocular regions (nasal, central and temporal). TMA was obtained by measuring the cross-sectional area of the inferior tear meniscus. Severity of conjunctivochalasis was quantified by measuring the conjunctivochalasis ratio (CCR), the ratio of area of redundant conjunctiva to the TMA. Ocular symptoms and demographic factors were assessed by standardized questionnaires. RESULTS: A total of 403 participants (52.9% women) 40 years of age and older were included. TMA centrally was 2818 ± 5308 pixel2. Female sex and the presence of meibomian gland dysfunction (MGD), but not older age, were associated with a lower TMA (p = 0.031, p = 0.031 and p = 0.956 respectively). In this population, 9.2% had severe conjunctivochalasis (CCR>0.7) whereas 39.0% had mild to no conjunctivochalasis (CCR≤0.3). Conjunctivochalasis was more severe in temporal, followed by nasal and central sections. Older age was associated with severe conjunctivochalasis (p < 0.001). CONCLUSION: MGD and female gender were associated with lower TMA, while older age was associated with increased severity of conjunctivochalasis. Objective measurement of TMA and CCR using ASOCT imaging may be useful in the assessment of tear volume and ocular surface tear function.

Hydrogen-peroxide and silicone-hydrogel contact lenses: Worsening of external eye condition and tear film instability.

PURPOSE: The aim is discussing the origins of worsening of external eye condition (EEC) and of tear film (TF) instability after wear of silicone-hydrogel contact lenses (CLs) with hydrogen-peroxide (H2O2) care system. METHODS: EEC and TF stability were evaluated before and after 15days of wear combined with different care systems: (1) H2O2, (2) detergent solution and H2O2, (3) multipurpose solution (MPS), (4) H2O2 and artificial tears. In-vitro cell mortality tests were performed after 24h cell incubation with CLs treated with H2O2. Photon correlation spectroscopy (PCS) was carried out on tears of non-wearers and CL wearers who used MPS or H2O2 solution. RESULTS: Worsening of EEC was observed only for the group using H2O2 (group 1). In-vitro, cell mortality was found higher for worn CL than for unworn CLs. Worsening of TF stability was observed regardless of care system and also PCS results on tears of CL wearers were found different compared to non-wearers regardless of care system. The only observed remedy for tear instability of CL wearers was found to be the administration of artificial tears. CONCLUSIONS: Worsening of EEC of CL wearers using H2O2 is attributed to H2O2 scarce cleaning efficacy, which can be solved by adding a CL detergent solution. The origin of TF instability is found to be different. A remedy was found to be the administration of artificial tears, whose effect could be attributed either to the role of specific components or to rinsing and replacement of TF during wear.

Use of Topical Cannabinomimetic Palmitoylethanolamide in Ocular Surface Disease Associated with Antiglaucoma Medications.

PURPOSE: Chronic use of topical hypotensive therapies in glaucoma patients leads to chronic inflammation of the ocular surface, which decreases the success rate of long-term glaucoma management. The aim of this study is to evaluate the effect of topical palmitoylethanolamide (PEA) (Defluxa©), a well-known anti-inflammatory and analgesic agent, in suppressing the ocular surface inflammation associated with the use of hypotensive eye drops. METHODS: In a pilot clinical trial, we enrolled 15 glaucomatous patients who received topical PEA (Defluxa) in addition to the current antiglaucoma drugs, while 15 glaucomatous patients did not receive any additional treatment. At 3 different time points (day 0, 15, and 30), signs of ocular surface involvement, adverse events, visual acuity, and intraocular pressure were assessed. RESULTS: Topical PEA (Defluxa) was effective in increasing the Schirmer test (P < 0.05) and the tear film breakup time (T-BUT) (P < 0.0001), and improving the conjunctival hyperemia (P < 0.0001) by day 30, compared to baseline. Compared to control, by day 15, the conjunctival hyperemia score was significantly decreased in the PEA (Defluxa) group (P < 0.01), while the T-BUT and the Schirmer Test achieved a significant improvement by day 30 (P < 0.05; P < 0.01). DISCUSSION: Our data suggests that topical PEA (Defluxa) is a safe, effective, and generally well-tolerated treatment to prevent or suppress ocular surface inflammation attributable to chronic glaucoma treatment.