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Purpose: This study aimed to compare the expression of vitamin D receptor (VDR) on the ocular surface of patients with dry eye disease (DED) and controls without ophthalmological pathologies., Methods: Patients with DED without previous treatment were studied and compared with healthy subjects. Ocular Surface Disease Index assessement and ocular surface cytology were performed in all patients. The immunohistochemical expression of VDR was evaluated using fully automated immunohistochemistry. The evaluation involved multiplying the percentage of nuclear-labeled cells (0-100) by their intensity (0-3), resulting in a score ranging from 0 to 300 (VDR H-score). Squamous metaplasia was morphologically evaluated using liquid-phase cytology with Papanicolaou/periodic acid-Schiff staining using Nelson's grade (scale 0-3, higher grade to higher metaplastic change)., Results: Eighteen patients with DED without previous treatment and 10 healthy subjects were studied. Squamous metaplasia was observed in 74% of patients with DED, in contrast to 0% in the control group. In patients with DED, there was a lower expression of VDR than in the control group (VDR H-score: 11.2 ± 13.9 vs. 80.9 ± 56; P = 0.0001). Furthermore, an inverse correlation was observed between Nelson grade and VDR H-score ( P = 0.0001, rs = -0.71). No correlation was observed between Ocular Surface Disease Index and VDR H-score., Conclusions: This is the first study to evaluate the VDR in patients with DED. These patients presented with a lower expression of VDR than healthy subjects. No correlation was found with more severe symptoms. Patients with DED also had a higher frequency of squamous metaplasia., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated pathogen., Methods: The host conjunctival transcriptome of 9 patients with Acanthamoeba keratitis (AK) is compared with the host conjunctival transcriptome of 13 patients with pathogen-free keratitis. Culture and/or confocal confirmed Acanthamoeba in 8 of 9 participants with AK who underwent metagenomic RNA sequencing as the likely pathogen. Cultures were negative in all 13 cases where metagenomic RNA sequencing did not identify a pathogen., Results: Transcriptome analysis identified 36 genes differently expressed between patients with AK and patients with presumed sterile, or pathogen-free, keratitis. Gene enrichment analysis revealed that some of these genes participate in several biologic pathways important for cellular signaling, ion transport and homeostasis, glucose transport, and mitochondrial metabolism. Notable relatively differentially expressed genes with potential relevance to Acanthamoeba infection included CPS1 , SLC35B4 , STEAP2 , ATP2B2 , NMNAT3 , and AKAP12 ., Conclusions: This research suggests that the local transcriptome in Acanthamoeba keratitis may be sufficiently robust to be detected in the conjunctiva and that corneas infected with Acanthamoeba may be distinguished from the inflamed cornea where no pathogen was identified. Given the low sensitivity for corneal cultures, identification of differentially expressed genes may serve as a suggestive transcriptional signature allowing for a complementary diagnostic technique to identify this blinding parasite. Knowledge of differentially expressed genes may also direct investigation of disease pathophysiology and suggest novel pathways for therapeutic targets., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: The purpose of this study was to assess the safety, tolerability, dosing, and efficacy of the active 19 amino acid fragment of lacritin (Lacripep), a broad regulator of ocular surface homeostasis, in the treatment of ocular surface disease associated with primary Sjögren syndrome., Methods: Two hundred four subjects were randomized to receive vehicle, 22 μM Lacripep, or 44 μM Lacripep 3 times daily for 28 days, preceded by a 14-day run-in and followed by 14-day washout. Outcome measures were corneal fluorescein staining (CFS), lissamine conjunctival staining, Schirmer with anesthesia, tear break-up time, SANDE scoring, and visual analog scale assessment of symptoms., Results: This study established the safety and tolerability of topical treatment with Lacripep in patients with primary Sjögren syndrome. There were few adverse events: Only mild irritation was found in less than 3 percent of patients dosed with Lacripep. Total CFS and Eye Dryness Score were not significantly changed at day 28. Post hoc analysis of patients with Eye Dryness Severity scores of 60 or greater at baseline revealed significant improvements in inferior CFS at 14 and 28 days and complaints of burning and stinging at 14 days. Significant improvement in regional lissamine conjunctival staining was seen at 14 and 28 days., Conclusions: This first-in-human study of Lacripep in patients with primary Sjögren syndrome demonstrated clinically significant improvements in specific signs and symptoms on which to base future studies. This study established safety and tolerability and potential metrics of efficacy in patients with moderate to severe disease. Further work on appropriate dosing and concentration is ongoing., Competing Interests: G. W. Laurie: Cofounder and CSO of TearSolutions, Inc; Financial interest in TearSolutions, Inc. E. C. Parsons: Consultant to TearSolutions, Inc. MGO: CMO of Tear Solutions, Inc; Financial interest in TearSolutions, Inc. The remaining authors have no funding or conflicts of interest to disclose., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: We investigated ocular surface microbiota dysbiosis in patients with refractory allergic conjunctival diseases (ACDs; stratified into mild and severe groups) treated with topical tacrolimus., Methods: Patients (n = 21) with refractory ACDs (including vernal and atopic keratoconjunctivitis) actively treated with topical tacrolimus and 6 healthy controls were evaluated. Based on clinical scores and expression of specific cytokines on the ocular surface, patients with ACDs were divided into mild and severe groups using cluster analysis. The microbial composition of tear specimens collected from patients with mild and severe ACD and control subjects using the Schirmer test paper was determined through next-generation 16S rRNA sequencing analysis., Results: Compared with healthy controls, patients with ACDs exhibited significantly decreased ocular surface microbiota α-diversity. Ocular surface microbiota mainly comprised members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria in all groups. The relative abundance of ocular surface microbiota in patients with ACDs was increased for phylum Firmicutes and decreased for phylum Proteobacteria (compared with control subjects). The genera Blautia (vs. mild ACD group) and Morganella (vs. control group) exhibited significantly increased abundance only in the severe ACD group., Conclusions: The ocular surface microbiota in patients with severe ACD exhibited decreased diversity and exacerbation of dysbiosis compared with that in patients with mild ACD and control subjects. Patients with mild refractory ACD also exhibited decreased diversity of these microbiota. These alterations in microbiota indicated a change in the ocular surface of patients with refractory ACD (be it because of disease pathogenesis or topical immunomodulatory treatment)., Competing Interests: J. Shoji received honoraria from Santen Pharmaceutical Co., Ltd. and Senju Pharmaceutical Co., Ltd. G. Harata and F. He are employees of Takanashi Milk Products Co., Ltd. The remaining authors have no conflicts of interest to disclose., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: To assess the efficacy, safety, and tolerability of a topical water-free cyclosporine A formulation (CyclASol 0.1% ophthalmic solution) in comparison with vehicle for the treatment of dry eye disease (DED)., Methods: Three hundred twenty-eight patients were enrolled in this prospective, 12-week, multicenter, randomized, double-masked, confirmatory, vehicle-controlled clinical study. After a 2-week run-in period, eligible DED patients were randomized 1:1 to either CyclASol 0.1% or vehicle twice daily. The primary efficacy endpoint was change from baseline in total corneal fluorescein staining (National Eye Institute scale), and the second hierarchical primary efficacy endpoint was change from baseline in the Ocular Surface Disease Index score, both at 4 weeks. Secondary efficacy and safety assessments included conjunctival lissamine green staining (Oxford scale), visual analog scales for dry eye symptoms, and adverse event., Results: Treatment with CyclASol 0.1% was superior to vehicle in the primary endpoint: total corneal fluorescein staining at week 4 (Δ -0.8; 95% confidence interval, -1.3 to -0.4; P = 0.0002, analysis of covariance). This difference had already reached statistical significance after 2 weeks and was maintained throughout the study. The study did not statistically meet its second hierarchically tested primary endpoint: Ocular Surface Disease Index score (P = 0.2634). However, CyclASol 0.1% treatment showed statistically significant improvement compared with that of vehicle in the eye dryness score at week 4 (Δ -4.783; 95% confidence interval, -9.129 to -0.438; P = 0.0311)., Conclusions: CyclASol 0.1% was effective in treating signs and symptoms of DED. It significantly reduced corneal and conjunctival staining and improved ocular dryness compared with vehicle. CyclASol 0.1% was safe and showed excellent tolerability., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: To confirm the ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by evaluating the expression of viral entry factors in human ocular tissues using immunohistochemistry., Methods: Fresh donor corneas and primary explant cultures of corneal, limbal, and conjunctival epithelial cells were evaluated for the expression of viral entry factors. Using immunohistochemistry, the samples were tested for the expression of angiotension-converting enzyme 2 (ACE2), dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), DC-SIGN-related protein (DC-SIGNR), and transmembrane serine protease 2 (TMPRSS2)., Results: In total, 5 donor corneas were evaluated for the expression of viral entry factors. In all specimens, both ACE2 and TMPRSS2 were expressed throughout the surface epithelium (corneal, limbal, and conjunctival) and corneal endothelium. In corneal stromal cells, ACE2 was sporadically expressed, whereas TMPRSS2 was absent. DC-SIGN/DC-SIGNR expression varied between donor specimens. Four specimens expressed DC-SIGN/DC-SIGNR in a similar distribution to ACE2, but 1 specimen from a young donor showed no expression of DC-SIGN/DC-SIGNR. ACE2, TMPRSS2, and DC-SIGN/DC-SIGNR were all expressed in the cultured corneal, limbal, and conjunctival epithelial cells., Conclusions: Both corneal and conjunctival epithelia express ACE2, DC-SIGN/DC-SIGNR, and TMPRSS2, suggesting that the ocular surface is a potential route for the transmission of SARS-CoV-2. The risk of viral transmission with corneal transplantation cannot be ruled out, given the presence of ACE2 in corneal epithelium and endothelium. Cultured corneal, limbal, and conjunctival epithelial cells mimic the expression of viral entry factors in fresh donor tissue and may be useful for future in vitro SARS-CoV-2 infection studies.

Purpose: Our eyes are chronically exposed to airborne particulate matter shown to adversely affect the ocular surface. This research examines size, type (organic vs. inorganic), and elemental composition of particles recovered from the ocular surface in 2 environments and their associations with dry eye (DE) metrics., Methods: Particles were recovered from the right eye using Schirmer strips obtained both in the clinic and home environments 9 ± 8 days apart. Particle size and elemental composition were assessed using scanning electron microscopy and energy dispersive spectroscopy. The paired t test was used to evaluate the differences in the size and types of ocular surface particles recovered from the clinic and home settings. Associations of particle size and type with home environmental conditions and DE measures were evaluated using correlation analyses., Results: The mean age of the 15 patients was 56 years, standard deviation (±) 12 years; 93% were men and 53% self-identified as white. Size, type, and elemental composition did not vary significantly between clinic and home. Particle surface area was marginally associated with home indoor temperature (25 °C ± 2, ρ=-0.53, P = 0.06) and significantly associated with the select DE signs: tear osmolality (304 mOsm/L ± 14, ρ= -0.60, P = 0.02), inflammation (0.7 ± 0.8, ρ = 0.53, P = 0.04), and tear breakup time (7 seconds ± 3, ρ = 0.56, P = 0.03)., Conclusions: Ocular surface particles were consistently detected across 2 different environments. Greater particle area detected on Schirmer strips correlated with some DE measures, suggesting that particles detected on the ocular surface may affect eye health.

Purpose: To investigate the expression levels of inflammatory cytokines in the conjunctival epithelium and correlations with clinical parameters in dry eye disease (DED)., Methods: This study evaluated 28 patients with Sjögren syndrome (SS) DED, 28 patients with non-SS DED, and 10 controls. The messenger ribonucleic acid (mRNA) expression of inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-17, and matrix metalloproteinase 9 (MMP9) from conjunctival epithelium was investigated by real-time polymerase chain reaction. Protein expression was confirmed by immunofluorescence staining. Correlations were evaluated between the mRNA expression of inflammatory cytokines and clinical DED parameters such as ocular surface disease index score, Schirmer I value, tear film breakup time, and corneal and conjunctival staining scores., Results: Patients with non-SS DED expressed significantly more IFN-γ, IL-6, and MMP9 genes in the conjunctival epithelium than the controls (P < 0.05), and all cytokine gene expression was significantly higher in patients with SS DED than in the controls (P < 0.01). Tumor necrosis factor-α, IL-1β, IL-6, and IL-17 gene expression was higher in patients with SS DED than in the non-SS DED group (P < 0.05). Immunofluorescence staining of conjunctival epithelium demonstrated that positive cells with IL-6 or MMP9 were significantly higher in non-SS DED than in controls (P < 0.01) and much higher in SS DED than in non-SS DED (P < 0.05). Conjunctival staining scores significantly correlated with the expression of IFN-γ, IL-6, IL-17, and MMP9 in both DED groups (P < 0.05 in non-SS DED and P < 0.01 in SS-DED). Interestingly, correlation coefficients of all cytokines were much higher in SS DED compared to non-SS DED. Corneal staining scores showed positive correlations with IFN-γ, IL-17, and MMP9 (P < 0.05), and correlation coefficients were lower than those of conjunctival staining scores., Conclusions: Conjunctival staining scores may be useful to measure ocular surface inflammation in SS and non-SS DED.

Purpose: To investigate the expression of thymic stromal lymphopoietin (TSLP) and the relevant signaling pathways in the giant papillae obtained from patients with vernal keratoconjunctivitis (VKC) and to study the potential functional role and molecular mechanism of TSLP., Methods: Giant papillae from VKC patients and control samples were used to perform immunohistochemical staining and analyze the mRNA expression of TSLP and related pathway by real-time polymerase chain reaction., Results: TSLP was markedly expressed in the epithelial cells and some inflammatory cells of giant papillae, but not in the control conjunctival tissue. TSLP mRNA expression in the giant papillae of VKC was increased by 9.63 ± 0.99 (mean ± SD) fold compared with controls (P < 0.01). CD11c and OX40L immunoreactive cells largely infiltrated the giant papillae as observed by immunohistochemical staining. CD4Th2 cell infiltration was observed through high immunoreactivity of CD4. Th2 cytokines (IL-4, IL-5 and IL-13) and OX40 in the VKC specimens showed increased expression. Augmented gene expression levels of CD4 (6.88 ± 1.84), OX40L (7.60 ± 1.79), OX40 (7.25 ± 1.38), IL-4 (6.89 ± 1.46), IL-5 (8.42 ± 1.55), and IL-13 (9.69 ± 1.94) were significantly different from controls (P < 0.05)., Conclusions: Our observations provide strong evidence that TSLP may be a crucial factor that contributes to the development and progression of allergic conjunctivitis. The results also demonstrated that TSLP activates dendritic cells to prime CD4T cells to differentiate into Th2 type and triggers Th2-dominant allergic inflammation through the TSLP/OX40L/OX40 signaling as part of immunopathogenesis of VKC.

Purpose: To describe an impression cytology (IC) technique using a purpose-built, sterile, EYEPRIM IC device that can be coupled with a TRIzol reagent-based RNA extraction protocol to yield sufficient RNA for gene expression analysis., Methods: IC samples using the EYEPRIM device were collected from the bulbar conjunctiva, with and without topical anesthesia, and evaluated for RNA yield, the absence of polymerase chain reaction (PCR) inhibitors, and the ability to detect biomarkers by quantitative real-time PCR and droplet digital PCR. A technique for collecting IC samples in the clinic, while preserving RNA, and a protocol for subsequent laboratory analysis of RNA were developed., Results: The extracted RNA was free of PCR inhibitors and could be synthesized into complementary DNA and used for successful relative quantification of ocular surface biomarkers by quantitative real-time PCR. For gene targets present in low abundance, complementary DNA could also be used for quantification by the relatively new and emerging method of droplet digital PCR. The described method was successfully used to evaluate 3 biomarkers in a clinical trial assessing the tolerability of a proprietary eyelid therapy in 92 IC samples from a study population of 46 participants., Conclusions: IC is a recognized technique for ocular surface cell evaluation and protein biomarker quantification but is infrequently used for quantifying gene expression. The EYEPRIM device allows ease of use and impression-to-impression consistency while accurate gene expression data offers a highly specific and sensitive method of disease characterization for clinician scientists to use in diagnosis.

Purpose: To evaluate the efficacy of intense pulsed light (IPL) therapy combined with meibomian gland expression (MGX) for refractory meibomian gland dysfunction (MGD) in a prospective study conducted at 3 sites in Japan., Methods: Patients with refractory obstructive MGD were enrolled and underwent 4 to 8 IPL-MGX treatment sessions at 3-week intervals. Clinical assessment included the Standard Patient Evaluation of Eye Dryness questionnaire; noninvasive breakup time of the tear film and interferometric fringe pattern as determined by tear interferometry; lid margin abnormalities, fluorescein breakup time of the tear film, corneal and conjunctival fluorescein staining (CFS), and meibum grade as evaluated with a slit-lamp microscope; meibomian gland morphology (meiboscore); and tear production as measured by the Schirmer test without anesthesia., Results: Sixty-two eyes of 31 patients (17 women, 14 men; mean age ± SD, 47.6 ± 16.8 years) were enrolled. The Standard Patient Evaluation of Eye Dryness score (P < 0.001), noninvasive breakup time (P < 0.001), and interferometric fringe pattern (P < 0.001) were significantly improved after therapy, with 74% of eyes showing a change in the interferometric fringe pattern from 1 characteristic of lipid deficiency to the normal condition. Meibum grade, lid margin abnormality scores, fluorescein breakup time, and CFS were also significantly improved (P < 0.001, P < 0.001, P < 0.001, and P = 0.002, respectively) after treatment, whereas the meiboscore and Schirmer test value remained unchanged., Conclusions: IPL-MGX ameliorated symptoms and improved the condition of the tear film in patients with refractory MGD and is therefore a promising treatment option for this disorder.

Purpose: To analyze the distribution of tear film osmolarity in patients with dry eye and its association with other ocular surface parameters., Methods: Tear osmolarity and other quantitative dry eye parameters were obtained from patients with 1) clinically significant dry eye (significant symptoms and ocular surface staining, n = 131), 2) symptoms-only dry eye (significant symptoms but no significant ocular surface staining, n = 52), and 3) controls (no significant symptoms or staining, n = 42)., Results: Tear osmolarity varied significantly across groups (P = 0.01), with patients with clinically significant dry eye having the highest tear osmolarity (312.0 ± 16.9 mOsm/L), control patients having the lowest tear osmolarity (305.6 ± 9.7 mOsm/L), and patients with symptoms-only dry eye falling in between (307.4 ± 5.6 mOsm/L). Patients with clinically significant dry eye also tended to have a greater intereye difference in osmolarity (12.0 ± 13.4) than did the individuals with symptoms-only dry eye (9.1 ± 12.4) and controls (9.0 ± 7.4) (P = 0.06). In multivariable regression models, higher tear osmolarity was associated with higher Ocular Surface Disease Index, discomfort subscore (P = 0.02), and higher corneal and conjunctival staining scores (P < 0.01 for both). Worse eye tear osmolarity was not correlated with the corresponding tear film breakup time or Schirmer test (P > 0.05 for both)., Conclusions: Individuals with symptomatic dry eye that is not yet clinically significant seem to have higher and more variable osmolarity measurements than controls, potentially indicating that changes in osmolarity precede clinical findings.

Purpose: The expression of peroxiredoxin 2 and vascular endothelial growth factor receptor 2 (VEGFR2) was detected in pterygium to investigate whether they are involved in the pathogenesis or recurrence of pterygium and to evaluate the association between peroxiredoxin 2 and VEGFR2 in pterygium., Methods: Ten normal bulbar conjunctivae, 35 primary pterygia, and 35 recurrent pterygia were obtained. Formalin-fixed, paraffin-wax-embedded tissues were analyzed by immunohistochemistry with peroxiredoxin 2 and VEGFR2 antibodies., Results: There was no statistical difference between primary pterygia and recurrent pterygia in terms of age and sex (P = 0.685; P = 0.811). The expression rate of peroxiredoxin 2 (94.3%, 66/70) and VEGFR2 (61.4%, 43/70) was increased in pterygia compared with normal conjunctivae (negative). The expression of peroxiredoxin 2 in recurrent pterygia (negative 0, weak 0, moderate 27, strong 8) was higher than that in primary pterygia (negative 6, weak 16, moderate 13, strong 0) (P < 0.001). The expression of VEGFR2 in recurrent pterygia (negative 4, weak 5, moderate 12, strong 4) was higher than that in primary pterygia (negative 23, weak 10, moderate 1, strong 1) (P < 0.001). The expression of peroxiredoxin 2 was consistent with that of VEGFR2 in pterygium (r = 0.348, P = 0.006)., Conclusions: Overexpression of peroxiredoxin 2 and VEGFR2 in pterygium might be involved in the pathogenesis or recurrence of pterygium. The increase of VEGFR2 might be related to the increase of peroxiredoxin 2 in response to excessive reactive oxygen species from ultraviolet exposure.

Purpose: To compare the therapeutic effects of topical tumor necrosis factor (TNF)-α-stimulated gene/protein-6 (TSG-6) with those of cyclosporine and prednisolone eye drops in NOD.B10.H2 mice, a model for inflammation-mediated dry eye., Methods: The 12-week-old NOD.B10.H2 mice were topically administered recombinant TSG-6 (0.1%) 4 times a day, 0.05% cyclosporine (Restasis) twice a day, or 1% prednisolone (Pred Forte) 4 times a day for 1 week. Aqueous tear production was measured by phenol red thread test, and corneal epithelial damage was observed with lissamine green and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Conjunctival goblet cell number was evaluated with periodic acid-Schiff staining. The levels of inflammatory cytokines were analyzed in the ocular surface (cornea and conjunctiva) and intraorbital gland. The dose-dependent effects of topical TSG-6 (0.001, 0.01, and 0.1%) were tested., Results: Tear production and goblet cell density were significantly increased in all groups receiving TSG-6, cyclosporine, and prednisolone. Corneal epithelial staining was markedly reduced by TSG-6 and cyclosporine but not by prednisolone. In prednisolone-treated eyes, corneal epithelial thickness was decreased, and apoptosis of corneal epithelial cells was increased. The levels of interferon gamma and TNF-α in the ocular surface and intraorbital gland were significantly repressed by TSG-6 and cyclosporine, and prednisolone treatment significantly reduced the level of interferon gamma. The effects of TSG-6 on the ocular surface and tear production were dose dependent., Conclusions: Topical TSG-6 was as effective in inflammation-mediated dry eye as cyclosporine eye drops. Topical prednisolone suppressed inflammation but induced apoptosis in the corneal epithelium.

Purpose: To assess the effects of autologous serum eye drops on conjunctival expression of the mucin gene MUC5AC by means of quantitative reverse transcription polymerase chain reaction (RT-qPCR)., Methods: A prospective and comparative interventional case series study of 38 eyes of 19 patients with different ocular surface disorders was performed before and 6 weeks after the treatment with autologous serum eye drops. All patients underwent a complete ophthalmic assessment, including evaluation of the tear film, ocular surface exploration, conjunctival impression cytology (IC), and MUC5AC detection by reverse transcription polymerase chain reaction., Results: A total of 34 eyes were studied by IC and MUC5AC quantitative reverse transcription polymerase chain reaction before and after treatment with autologous serum. This treatment improved breakup time, conjunctival squamous metaplasia, goblet cell density, and subjective perception in 76.2%, 70.6%, 55.9% and 73.5% of eyes, respectively. Treatment with autologous serum enhanced conjunctival expression of MUC5AC (P = 0.001), although these differences were not statistically significant if data are analyzed patient by patient (P = 0.09). In 13 of 34 eyes (38.2%), we found increased expression of MUC5AC; in 12 eyes (35.3%), no significant changes were found; and in 9 eyes (26.5%), a decreased expression was found. The MUC5AC gene upregulation was related to the conjunctival involvement before treatment and with the improvement in the degree of squamous metaplasia and the increase in the number of goblet cells in IC after treatment (P = 0.001)., Conclusions: Treatment with autologous serum enhances the conjunctival expression of MUC5AC by increasing the density of goblet cells, mainly in patients with severe conjunctival involvement.

Purpose: Inflammation and squamous metaplasia is a common pathological process that occurs in many ocular surface diseases such as dry eye, Stevens-Johnson syndrome, mucous membrane pemphigoid, and chemical/thermal burns. At present, there is no ideal medicinal treatment for this abnormality. We report herein on an ex vivo conjunctival inflammation and squamous metaplasia model by culturing human conjunctival tissues at an air-liquid interface for up to 8 days to study the effects of minocycline on inflammation and squamous metaplasia., Methods: The levels of inflammatory mediators including interleukin-1β, tumor necrosis factor-α, and metalloproteinase-9 in the cultured human conjunctival tissues were determined by enzyme-linked immuno-sorbent assay and real-time polymerase chain reaction. The total and phosphorylated nuclear factor-κB were detected by western blot. Differentiation markers K10, MUC5AC, and Pax6 and proliferation markers Ki67, p63, and K14 were determined by immunofluorescence or immunohistochemical staining., Results: The results indicated that minocycline inhibited inflammation, decreased the expression of interleukin-1β, tumor necrosis factor-α, and metalloproteinase -9, and squamous metaplasia features such as hyperproliferation and abnormal epidermal differentiation of conjunctival epithelium., Conclusions: These findings highlight the possibility that minocycline could be used to treat dry eye and other ocular surface diseases exhibiting epithelial cell inflammation and squamous metaplasia.

Purpose: To investigate the differential expression of stem cell factor (SCF) and thymic stromal lymphopoietin (TSLP) and their correlation to mast cells, between patients diagnosed with superior limbic keratoconjunctivitis (SLK) and normal subjects., Methods: A laboratory investigation included 22 surgical specimens of the superior bulbar conjunctiva from 17 patients with medically refractory SLK and 5 control subjects who underwent cataract or retinal surgery. Protein expression of tryptase, SCF, and TSLP in conjunctival specimens was detected by immunohistochemistry. The number of mast cells was correlated with immunohistochemistry intensity of SCF and TSLP., Results: In patients with SLK, higher immunostaining intensity of SCF and TSLP was found in the conjunctival epithelium than that in the conjunctival subepithelial stroma. SCF and TSLP staining in the conjunctival epithelium was significantly more intense in patients with SLK than in normal subjects. In addition, there was a significant correlation between the number of tryptase (+) mast cells in conjunctival subepithelial stroma and TSLP immunointensity in the conjunctival epithelium and subepithelial stroma., Conclusions: Overexpression of SCF and TSLP was found in the conjunctival epithelium of patients with SLK. Significant correlation between TSLP grading and the number of mast cells was also found. SCF and TSLP may be involved in promoting mast cell migration and activation contributing to the pathogenesis of SLK.

Purpose: To evaluate and compare corneal and conjunctival biomarkers for immunocytochemical diagnosis of limbal stem cell deficiency (LSCD)., Methods: In accordance with the current literature, we selected K12 as the corneal biomarker and K7/K13/K19/MUC5AC as the conjunctival ones. The specificity and accuracy for each biomarker were assessed and compared on 10 healthy subjects and tissues of deceased donors. Twelve eyes of 9 patients clinically suspected of LSCD were enrolled. Epithelial cells (ECs) from the central cornea were collected using impression cytology (IC) and assessed for each biomarker. The presence of conjunctival cells in the central cornea was diagnostic proof of LSCD, whereas the detection of corneal residual cells would quantify the degree of LSCD., Results: K12 and K7/K13/MUC5AC are, respectively, highly specific of corneal and conjunctival differentiation, whereas K19 is not. Normal corneal ECs are not desquamative enough to be suitable for IC. Among 12 eyes with suspected LSCD, 84% (10 of 12) of IC samples were suitable for analysis. K3/K7/K19 immunostaining was positive in 100%, MUC5AC in 40%, and K12 was never observed., Conclusions: Clinical examination can lead to misdiagnosis of LSCD. Immunocytochemical detection of K7/K13 on corneal ECs collected by IC is reproducible, noninvasive, and highly effective in this indication, but without any quantification of the degree of the disease. This time-consuming technique requires skilled technicians and laboratory facilities, reserving it for planned limbal reconstruction.

Purpose: IL-21 plays an important role in primary Sjögren syndrome (SS) pathogenesis. The purpose of this study was to evaluate IL-21 expression in tears and the conjunctiva and to analyze the impact of IL-21 on primary SS dry eyes., Methods: Eighty subjects were enrolled in this study: 30 patients with primary SS dry eye (30 eyes); 30 patients with non-SS dry eye (30 eyes), and 20 normal controls. Tear IL-21 levels were measured by flow cytometry, and IL-21 gene expression in the conjunctiva from impression cytology was evaluated by quantitative polymerase chain reaction. Ocular Surface Disease Index, tear film breakup time, Schirmer I test, and ocular surface staining scores were obtained for all patients., Results: Primary SS dry eyes had significantly higher tear IL-21 levels than non-SS dry eyes and normal controls (P < 0.01). In addition, IL-21 gene expression in the conjunctiva was also higher in primary SS dry eyes than in non-SS dry eyes and normal controls (P < 0.01). However, there were no significant differences in IL-21 expression in tears and the conjunctiva between non-SS dry eyes and controls. The tear IL-21 level was significantly correlated with ocular surface stain scores (r = 0.54, P < 0.01) and Schirmer I test values (r = -0.23, P < 0.05) in primary SS dry eyes., Conclusions: Our findings suggest that severity of primary SS dry eye is associated with IL-21.

Purpose: The aim of this study was to evaluate tear film osmolarity in patients with ocular mucous membrane pemphigoid (MMP)., Methods: This observational cross-sectional study included 40 patients with biopsy-proven ocular MMP at Foster stage III referred to the tertiary-care Ocular Immunology and Uveitis Service at the San Raffaele Scientific Institute in Milan from June 2010 to August 2013. We evaluated the following clinical parameters: tear film osmolarity, ocular surface disease symptoms (OSDI) questionnaire, Schirmer test, tear film break-up time (TFBUT), and corneal and conjunctival staining., Results: Forty patients (27 women and 13 men) were enrolled. All patients were undergoing systemic immunosuppressive therapy: 19 patients (47.5%) were on methotrexate, 9 (22.5%) were on mycophenolate mofetil, 9 (22.5%) were on low-dose corticosteroids, and 3 (7.5%) were on azathioprine. The mean osmolarity was 322.90 ± 33.39 mOsm/L, the mean OSDI score was 73.2 ± 17.9, the mean TFBUT was 6.60 ± 3.13 seconds, and the mean Schirmer test value was 4.07 ± 3.58 seconds. Tear film osmolarity significantly correlated with the TFBUT (r = 0.80; P < 0.0001), whereas no clinical correlation was found with the Schirmer test value (r = 0.01; P = 0.40) or with the OSDI score (r = 0.02; P = 0.29). Osmolarity did not turn out to be statistically different in the subgroups according to the Oxford corneal staining scale (P = 0.71) and to the Van Bijsterveld conjunctival staining score (P = 0.31)., Conclusions: Tear osmolarity increased in patients with ocular MMP and correlated with the TFBUT. This result emphasizes the role of evaporative dry-eye condition in patients with ocular MMP. Tear osmolarity may be considered as a useful test in the diagnostic assessment of dry eye associated with MMP and for targeting therapeutic decisions.

Purpose: We evaluated light exposure-induced dry eye syndrome by investigating the phototoxic effects of an operating microscope on the ocular surface and tear film in rabbits., Methods: Sixty eyes of 30 rabbits were divided into 3 groups based on the intensity of light exposure received from an operating microscope: Control group, no exposure to light; group A, 40,000-lx intensity for 30 minutes; and group B, 100,000-lx intensity for 30 minutes. To evaluate the potential damage to the ocular surface and tear film, Schirmer tests, rose bengal staining, and conjunctival impression cytology were performed before the light exposure and at 1, 3, and 5 days afterward. In addition, the expression of interleukin 1-beta was analyzed in tear samples. The expression of mucin 5AC was evaluated using immunofluorescence staining, and periodic acid-Schiff staining was conducted on conjunctival tissues. Corneal and conjunctival tissues were observed by means of electron microscopy., Results: Potential damage to the ocular surface and tear film was found in the light-exposed groups as evidenced by decreased aqueous tear production, devitalized corneal and conjunctival epithelial cells, squamous metaplasia of conjunctival epithelial cells, decreased conjunctival goblet cell density, decreased expression of mucin 5AC, ultrastructural cellular damage to corneal and conjunctival tissues, and increased interleukin 1-beta expression in tears. This damage was more noticeable in group B than in group A (P < 0.05)., Conclusions: Light exposure from an operating microscope had phototoxic effects on the ocular surface and tear film in this in vivo experiment. These changes seemed to intensify as the intensity of the light increased. Therefore, excessive light exposure during ophthalmic procedures could be a pathogenic factor in dry eye syndrome after a surgery is performed.

Purpose: Recent studies have shown that aquaporins (AQPs) play an important role in proliferating tumor microvessels and angiogenesis. In this study, the authors investigated the expression of aquaporin-1 (AQP1) and aquaporin-3 (AQP3) in pterygial and normal conjunctival tissues., Methods: Fifteen patients with pterygium were enrolled in the study. Pterygium was excised, and a conjunctival rotational flap or autograft was inserted. Normal conjunctival tissue was obtained from the flap or graft. Western blot analysis was performed to assess the expression of AQP1 and AQP3 in pterygial and normal conjunctival tissues. Tissue localization of AQP1 and AQP3 was determined by immunohistochemical analysis., Results: AQP1 and AQP3 are localized in the epithelial and subepithelial regions in pterygial and normal conjunctival tissues. Protein expression of both AQP1 and AQP3 was elevated in pterygia when compared with conjunctival tissues. The significant increase in protein expression of AQP1 was 3-fold in pterygium over normal conjunctiva (P = 0.004) and 2-fold increase in AQP3 expression of pterygium was detected (P = 0.02) according to densitometric analysis., Conclusions: Elevated protein expression of AQP1 and AQP3 was observed in pterygial tissues when compared with normal conjunctiva. The data suggest that the increased expression of AQP1 and AQP3 in pterygial tissues may be involved in the pathogenesis of pterygia, and therefore, AQP1 and AQP3 are potential therapeutic targets for preventing or delaying the progression of the disease.

Purpose: To investigate the expression of mucin and inflammatory factors in the conjunctival epithelium of subjects with dry eye and determine a correlation between clinical laboratory tests and subjective symptoms., Methods: The ocular surface disease index questionnaire, corneal fluorescein staining, tear film breakup time, and Schirmer I tests were administered in 66 subjects. According to questionnaire scores, the subjects were divided into mild, moderate, and severe symptom groups. Then, the subjects were divided based on the clinical tests into negative and positive groups. Impression cytology specimens from all patients were immunolabeled with antibodies directed against human mucin 5AC (MUC5AC) and interleukin-6 (IL-6) and the staining density was measured., Results: MUC5AC was lower in the severe symptom group compared with the mild and moderate symptom groups (P < 0.001, respectively). IL-6 was higher in the severe symptom group compared with the mild and moderate symptom groups (P < 0.001, respectively), and also in the moderate symptom group compared with the mild symptom group (P < 0.001). A decrease of MUC5AC was found in the positive group compared with the negative group (P < 0.001). IL-6 was higher in the positive group compared with the negative group (P = 0.029). MUC5AC was negatively correlated with the ocular surface disease index scores (r = -0.697, P < 0.001), whereas IL-6 positively correlated the scores (r = 0.895, P < 0.001). There were weak correlations for MUC5AC and IL-6 with tear film breakup time (r = 0.158 and r = -0.195, respectively; P = 0.038 and P = 0.041, respectively), and no correlations with other clinical tests., Conclusions: Dry eye disease is associated with decreased levels of mucin and an overexpression of IL-6, which correlate to the symptomatic severity of disease.

Purpose: To compare the intraocular penetration of 4 fluoroquinolone eye drops after topical instillation into rabbit eyes., Methods: The tested drugs were levofloxacin 1.5% (LVFX), gatifloxacin 0.3%, moxifloxacin 0.5% (MFLX), and besifloxacin 0.6% (BFLX). Forty-eight New Zealand white rabbits were randomly assigned into 2 groups. For group 1 (40 rabbits, 80 eyes), single instillation was performed, and tissue samples were acquired after 0.5, 1, 2, 4, and 6 hours. For group 2 (8 rabbits, 16 eyes), repeated instillation was performed (4 times, every 15 minutes), and tissues were acquired 1 hour after the fourth instillation. The drug concentrations in ocular tissues (cornea, aqueous, conjunctiva, and trisected vitreous) were analyzed with high-performance liquid chromatography., Results: The AUC 0-6 h (area under the curve, in microgram.hour/gram) in group 1 and the mean concentration (in micrograms/gram) in group 2 for LVFX, gatifloxacin 0.3%, MFLX, and BFLX, respectively, were 22.97, 6.44, 13.54, and 3.29 and 22.60, 6.99, 13.69, and 1.91 in cornea; 5.66, 1.43, 3.38, and 0.42 and 5.52, 1.29, 2.47, and 0.19 in aqueous humor; 2.33, 0.91, 2.17, and 9.83 and 4.51, 0.78, 1.48, and 2.09 in bulbar conjunctiva; 0.243, 0.051, 0.134, and 0.018 and 0.182, 0.055, 0.122, and 0.015 in anterior vitreous; none of the drugs achieved enough concentration in equatorial and posterior vitreous. Repeated instillation resulted in approximately 2.1 times greater penetration than single instillation., Conclusions: LVFX and MFLX demonstrated good intraocular penetration particularly in cornea, aqueous humor, and anterior vitreous, and they may be considered the penetrative fluoroquinolones. BFLX showed high concentration in bulbar conjunctiva and may be considered the retentive fluoroquinolone.

Purpose: The aim of this study was to compare the ocular and systemic distribution of cyclosporine A (CsA) in rabbits after the instillation of preservative-free CsA cationic and anionic emulsions., Methods: For the single-dose pharmacokinetic (PK) study, rabbits were instilled with 50 μL of the test material. For the multiple-dose PK study, rabbits were instilled twice daily with Restasis or once daily with NOVA22007 for 10 days. At each time point, the cornea, conjunctiva, and whole blood were harvested for CsA quantification. Ocular and systemic distribution were determined after 4 times daily instillations with 50 μL of 3H-CsA cationic and anionic emulsions for 7 days. Restasis was used as a reference in all studies., Results: Single-dose PK data demonstrated that NOVA22007 0.1% and 0.05% delivered higher CsA concentrations to the cornea than Restasis [concentration maximum (C max): 2692, 1372, and 748 ng/g, respectively] and have a better exposition (area under the curve). Conjunctival Cmax values were 1914, 696, and 849 ng/g and area under the curve values were 3984, 2796, and 2515 ng/g · h, for either dose of the cationic emulsions and Restasis, respectively. The multiple-dose PK and the 3H-CsA distribution data demonstrated that the systemic distribution after repeated instillations was low and comparable for all emulsions., Conclusions: These data demonstrate that the CsA cationic emulsions were more effective than Restasis at delivering CsA to target tissues, thus confirming the potential advantage of cationic emulsions over anionic emulsions as vehicle for ocular drug delivery for the treatment of ocular surface diseases.

Purpose: Cationic emulsions (CEs), developed as vehicles for lipophilic drugs, have been shown to be safe and effective for the treatment of dry eye. The aim of this study was to investigate the effects of a preservative-free latanoprost 0.005% CE (latanoprost-CE) in in vitro and in vivo models of corneal wound healing., Methods: An in vitro wound was made by scraping through a confluent layer of human corneal epithelial cells. Cytotoxicity, cell migration, and proliferation were analyzed after an exposure to phosphate-buffered saline, CE, latanoprost-CE, 0.02% benzalkonium chloride (0.02%BAK), and Xalatan (latanoprost). In vivo, the recovery and integrity of corneal wound healing were assessed in rat eyes instilled twice a day for 5 days with the above treatments after deepithelialization of the superior cornea., Results: In vitro wound distances decreased at 2 and 24 hours for human corneal epithelial cells exposed to CE, latanoprost-CE, and phosphate-buffered saline, whereas they progressively increased for 0.02%BAK-treated and latanoprost-treated cells. The greater wound closure was associated with a higher number of Ki67-positive cells. In CE- and latanoprost-CE-treated rats, reepithelialization of the cornea was enhanced, restoring normal appearance and function. In contrast, 0.02%BAK or latanoprost delayed corneal healing, induced inflammation, and decreased MUC5-AC expression., Conclusions: Both models effectively evaluated the cytotoxicity and dynamic recovery of corneal wound healing, and their correlation supports the potential of the in vitro model as a reliable alternative to in vivo ocular toxicity tests. Both models demonstrated that in the face of corneal injury, CEs favored corneal healing, whereas BAK was deleterious.

Purpose: To investigate the efficacy of a topical anti-tumor necrosis factor-α agent, infliximab, in a mouse model of experimental dry eye (EDE)., Methods: EDE was induced in C57BL/6 mice, with or without topical treatment consisting of balanced salt solution or 0.001%, 0.01%, or 0.1% infliximab solutions. Tear volume and corneal smoothness were measured on days 5 and 10 after treatment. Levels of interleukin (IL)-1β, IL-6, IL-17, and interferon γ (IFN-γ) were measured in the conjunctiva using a multiplex immunobead assay 10 days after treatment. Periodic acid-Schiff staining, immunohistochemistry, and flow cytometry were also performed 10 days after treatment., Results: Mice treated with 0.01% or 0.1% infliximab showed a significant improvement in tear volume and corneal smoothness compared with controls. The 0.01% and 0.1% infliximab-treated groups showed decreased levels of conjunctival IL-1β, IL-6, IL-17, and interferon γ and a decreased staining intensity of tumor necrosis factor-α. The density of conjunctival goblet cells was higher, whereas the number of CD4*CXCR3* T cells was lower, in the 0.01% and 0.1% infliximab-treated groups compared with the EDE and balanced salt solution control groups. However, there was no significant difference in all parameters between the 0.001% infliximab-treated group and control group., Conclusions: : Topical application of infliximab can improve tear production and ocular surface irregularity, decrease inflammatory cytokines and cells on the ocular surface, and increase conjunctival goblet cell density. These results suggest that topical infliximab eye drops at a concentration of 0.01% and 0.1% may be useful for the treatment of dry eye disease.

Purpose: To investigate the effect of topical pranoprofen 0.1% on the clinical evaluation and conjunctival human leukocyte antigen II (HLA-DR) expression in dry eyes., Methods: Sixty patients with dry eyes were randomized to 2 groups. Patients in group 1 received topical pranoprofen 0.1% plus topical sodium hyaluronate 0.1%; and patients in group 2 received sodium hyaluronate without pranoprofen. Ocular surface disease index (OSDI), tear &filig;lm break-up time (TBUT), Schirmer I test, ocular surface staining (OSS), and conjunctival HLA-DR expression were evaluated before treatment and at 15 and 30 days after treatment., Results: On day 15, patients in group 1 had significantly lower OSDI, OSS, and HLA-DR-positive cells compared with patients in group 2 (P < 0.01), and the TBUT was significantly longer in patients in group 1 than that of patients in group 2 (P < 0.01). On day 30, the difference between the 2 groups in OSS lost significance; however, there continued to be significant differences in the OSDI, TBUT, and HLA-DR expression between the 2 groups (P < 0.01). On days 15 and 30, the values in group 1 patients had significant improvement compared with their baseline values in terms of the above-mentioned parameters. The comparisons within group 2 did not reveal any significant differences. There was no significant effect in the Schirmer I test value in eyes of patients in group 1 or group 2 at days 15 or 30 (P > 0.05)., Conclusions: Topical pranoprofen 0.1% has a beneficial effect in reducing the ocular signs and symptoms of dry eyes and decreasing the inflammatory markers of conjunctival epithelial cells.

Purpose: To evaluate the effect of subconjunctivally injected liposome-encapsulated tissue plasminogen activator (tPA) on the absorption rate of subconjunctival hemorrhages (SHs)., Methods: SHs were induced in 1 eye each of 36 rabbits by subconjunctival injection of 0.05 mL of autologous blood. After 8 hours, randomized subconjunctival injections were performed: 26,000 IU/mL liposome-encapsulated tPA (0.05 mL) in 9 eyes (group A), free-form tPA (26,000 IU/mL; 0.05 mL) in 9 eyes (group B), only liposomes (0.05 mL) in 9 eyes (group C), or no injection in 9 eyes (group D). The sizes of the SHs at 8, 24, 48, 72, 96, and 120 hours after induction were measured using an image analyzer and were compared among the 4 groups., Results: Group A showed significantly more rapid absorption rates than all the other groups at 24, 48, and 72 hours and had the significantly shortest mean elapsed time for the complete resorption of SHs. The tPA activity in ocular tissue except conjunctiva and plasma were negligible beyond 24 hours after SH induction with both forms of tPA. In the conjunctiva, the tPA activity was significantly prolonged in the liposome-encapsulated tPA group than in the free tPA group., Conclusions: Subconjunctival injection of liposome-encapsulated tPA seems to enhance SH absorption in rabbits, especially during the early stages with minimal systemic and ocular absorption.

Purpose: To determine if levels of the glycocalyx membrane mucins, MUC1 and MUC16, and the secreted goblet cell mucin MUC5AC are altered in conjunctival cells and tears of postmenopausal women presenting with a history of non-Sjögren dry eye and if mucin levels correlate with dry eye clinical diagnostic data., Methods: Eighty-four postmenopausal women with a history of non-Sjögren dry eye and 30 normal subjects were recruited for this study. Impression cytology samples were collected for mucin messenger RNA (mRNA) and protein analysis. Tears were collected for mucin protein assay. Quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to quantitate MUC1, MUC16, and MUC5AC levels., Results: Postmenopausal women with a history of dry eye displayed significantly increased MUC1 mRNA expression and cellular protein compared with normal subjects (P < 0.001 and P < 0.01, respectively). Similarly, cellular MUC16 protein levels were significantly higher (P < 0.001). Mucin levels were found to be correlated with the clinical characterization of the subjects, including staining and symptoms. Although cellular MUC5AC protein levels were increased in symptomatic subjects, the increase did not reach statistical significance., Conclusions: Elevation in MUC1 and MUC16 mRNA and/or protein levels in postmenopausal women with non-Sjögren dry eye with a history of dry eye may be a compensatory response to irritation and inflammation associated with the disease. Understanding the pattern of mucin expression associated with the dry eye pathology may clarify factors involved in the progression of the disease and enhance the development of targeted therapies.

We first investigated whether the prostaglandin (PG) E2-PGE receptor subtype EP3 axis regulates the development of murine experimental allergic conjunctivitis because it has been reported that this pathway negatively regulates allergic reactions in a murine allergic asthma model. We observed that EP3 is constitutively expressed in mice conjunctival epithelium. EP3 knockout mice demonstrated significantly increased eosinophil infiltration in conjunctiva after ragweed challenge compared with wild-type mice. Consistently, significantly higher expression of eotaxin-1 messenger RNA was observed in Ptger3-/- mice. Conversely, treatment of wild-type mice with an EP3-selective agonist significantly decreased eosinophil infiltration, which was blunted in Ptger3-/- mice. Expression of cyclooxygenase-2 and PGE synthases was upregulated and PGE2 content increased in the eyelids after ragweed challenge. These data suggest that PGE2 acts on EP3 in the conjunctival epithelium and downregulates the progression of experimental allergic conjunctivitis. We next examined and compared the expression of EP3 in human conjunctival epithelium in various ocular surface diseases. Human conjunctival epithelium expressed EP3-specific messenger RNA and EP3 protein. Although we could clearly find positive signals in the conjunctival epithelium from patients with noninflammatory ocular surface diseases such as conjunctivochalasis and pterygium, we could not find positive signals in that from those with inflammatory disorders such as Stevens-Johnson syndrome and ocular cicatricial pemphigoid. Likewise, expression of the PGE receptor subtype EP4 was clearly found in the conjunctival epithelium from patients with conjunctivochalasis and pterygium but not from patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid.

Backgrounds: We showed previously that costimulatory molecules such as OX40 and 4-1BB participate in the development of murine allergic conjunctivitis (AC). We investigated whether these molecules are expressed in the conjunctiva of patients with severe AC., Methods: Giant papillae were collected from 4 patients with severe AC. As a control, bulbar conjunctivas from 4 normal volunteers were used. Frozen sections of the conjunctivas were stained with anti-CD4, anti-CD8, anti-OX40, anti-4-1BB, or control antibodies (Abs) and then incubated with the biotinylated secondary Ab. Alternatively, staining was performed with a biotinylated anti inducible costimulator (ICOS) Ab. After using an avidin-biotin complex kit, the staining levels were graded from 0 to 3. For immunofluorescence analysis, conjunctival sections were stained with primary Abs that had been fluorescently labeled., Results: The conjunctival stroma of all 4 patients with severe AC were infiltrated with CD4+ and CD8+ cells, whereas only 1 control sample contained stromal CD4+ and CD8+ cells. Conjunctivas from patients but none of the control conjunctivas expressed OX40, 4-1BB, and ICOS. The expression levels of these molecules were significantly higher in patients than the control volunteers. Immunofluorescence analysis demonstrated that the OX40 and 4-1BB, but not the ICOS, molecules were expressed by the infiltrating CD4+ and/or CD8+ cells., Conclusions: Together with the data in murine AC, OX40 and 4-1BB are expressed in the conjunctiva of patients with severe AC but not in the control conjunctivas, which suggests that OX40 and 4-1BB participate, at least in part, in the development of severe AC.

Purpose: Cosmetic product application may be an etiologic risk factor for dry eye syndrome. This study aimed to investigate whether a cosmetic product material (CPM) could migrate onto the ocular surface and whether eyedrop instillation might exacerbate this migration., Methods: CPM was prepared by mixing equal volumes of hydroxyethyl cellulose gel (Scopisol; Senju Pharmaceutical Co. Ltd., Osaka, Japan) and a 10% fluorescein solution. Seventy-five female volunteers were randomly separated into three groups: the outer eyelash line group, in which CPM was applied on the upper and lower eyelids approximately 2 mm from the eyelash line; the eyelash line (EL) group, in which CPM was applied close to the eyelash line but without touching it; and the inner eyelash line group, in which CPM was applied on the eyelash line extending to the margin of the eyelids. The right eye was used for this study. At 5 and 30 minutes after CPM application, slit-lamp examination was carried out to detect fluorescence, an indication of CPM migration and contamination of the ocular surface. A strip of Schirmer test paper was used to collect tears, and the color of the paper was scored (contamination score: 0-5) to determine the relative quantity of CPM contamination on the ocular surface. In another set of experiments, 5 minutes after CPM application, one drop of balanced saline solution was instilled into the eye. Thirty seconds later, eyes were examined by slit-lamp microscopy and tears were collected to determine the migration percentage (eyes with CPM migration/total eyes) and contamination score., Results: At 5 minutes after CPM application, the inner eyelash line group displayed a significantly higher migration percentage (96%) and average contamination score (3.35 ± 0.77) compared with the outer eyelash line (12%; 0.15 ± 0.34) and EL (20%; 0.35 ± 0.32) groups. At 30 minutes, both the migration percentage and the average contamination score increased remarkably in the EL group (60% and 2.71 ± 1.01). The migration percentage and average contamination score in the outer eyelash line group (P = 0.005 and P = 0.003, respectively) and EL group (P = 0.009 and P = 0.006, respectively) were significantly higher after eyedrop instillation compared with values before eyedrop instillation., Conclusion: CPM can migrate onto the ocular surface when applied close to the eyelid margin. This migration increases with time and can be exacerbated by eyedrop instillation.

Purpose: To present a case of perilimbal pigmentation as a complication of cosmetic blepharopigmentation., Methods: Interventional case report., Results: A 54-year-old white woman underwent bilateral upper and lower eyelid cosmetic blepharopigmentation. After the procedure, the patient noted a small area of black pigmentation of the surface of her right eye. Slit-lamp examination revealed perilimbal pigmentation involving the conjunctiva and the superficial cornea of the right eye. The affected conjunctiva was excised, and a diamond burr used to remove the underlying pigmented superficial cornea and sclera. The excised conjunctiva was analyzed by standard light microscopy. Hematoxylin and eosin-stained sections of the surgical specimen showed a thin ribbon of pigmentation just under the conjunctival epithelium without obvious inflammatory cell reaction around the extracellular pigment. The patient tolerated the surgical removal of the pigmentation without any complications and was pleased with the cosmetic result., Conclusions: Possible complications of blepharopigmentation include pigment deposition into the conjunctiva and the superficial cornea and sclera as was seen in this case report. This case of inadvertent pigmentation did not cause any significant chronic inflammation, although acute and chronic inflammatory reactions of the conjunctiva and surrounding structures have been reported as complications after cosmetic blepharopigmentation. Surgical removal of the affected tissue is a treatment option and was successful in removing the pigment in this case.

Purpose: To investigate the distribution of vimentin-expressing cells in human pterygium by immunocytochemical analysis of impression cytology specimens., Methods: Using impression cytology, 3 samples of superficial conjunctival epithelial cells, including pterygium and adjacent conjunctiva were obtained from each of 24 eyes of 24 patients with unilateral primary pterygium, 16 eyes of 16 patients with unilateral recurrent pterygium, and the 40 unaffected fellow eyes. Specimens were processed in avidin-biotin complex immunostaining system with monoclonal anti-vimentin antibody and/or anti-CD1a antibody and counterstained with hematoxylin. Under light microscopy, the extent of vimentin-expressing cell infiltration beyond the pterygium margin was measured with a micrometer and the number of vimentin-expressing cells was counted in each of 6 randomly selected high-power fields in the specimens of pterygium and normal conjunctiva., Results: Two types of cells expressed vimentin, epithelioid cells and dendritic cells, whereas cells that expressed CD1a showed only a dendritic pattern. Vimentin-expressing round epithelioid cells (presumed pterygial cells) were present over the entire surface of the pterygium and also on adjacent normal-appearing conjunctiva, up to an average of 3.01 +/- 0.83 mm beyond the superior margin and 3.14 +/- 0.99 mm beyond the inferior margin of primary pterygium and 3.72 +/- 0.85 mm and 3.58 +/- 0.98 mm beyond the superior and inferior margins of recurrent pterygium. For the superior margin but not the inferior margin, the clinically occult extension of vimentin-expressing epithelioid cells was statistically significantly greater for eyes with recurrent pterygium compared with those with primary pterygium (P = 0.016). The density of dendritic (Langerhans) cells, stained by anti-vimentin and anti-CD1a antibodies, was statistically significantly lower in normal fellow eyes than in eyes with either primary (P < 0.001) or recurrent (P = 0.024) pterygium., Conclusions: The vimentin-expressing epithelioid cells were present not only over the ocular surface of the pterygium but also in the normal-appearing conjunctiva adjacent to primary and recurrent pterygium. The increased density of Langerhans cells in pterygium might reflect a higher level of antigenic and mitogenic exposure in the conjunctiva. However, the significance of these 2 phenomena in the recurrence and pathogenesis of pterygium remains undetermined and merits further studies.

Purpose: To evaluate the effects of sequential treatment with artificial tears and cyclosporine emulsion on conjunctival goblet cell density and production of transforming growth factor (TGF)-beta2 in patients with dry eye disease., Methods: Patients with dry eye disease (N = 6) defined by an Ocular Surface Disease Index symptom score >or=25, Schirmer test 1 <10 mm, and corneal fluorescein and conjunctival lissamine green staining scores >or=3 were treated with artificial tears (Refresh Plus; Allergan, Irvine, CA) 4 times a day for 4 weeks, followed by 0.05% cyclosporine emulsion (Restasis; Allergan) twice a day for 12 weeks. Impression cytology was performed on the bulbar conjunctiva of both eyes at baseline, after artificial tear therapy, and after 6 and 12 weeks of cyclosporine therapy. Goblet cells were counted in 5 representative microscopic fields per membrane in those taken from the temporal and inferior bulbar conjunctiva of the worse eye, and membranes taken from the fellow eye were immunostained for TGF-beta2., Results: There were no differences in mean goblet cell density between baseline and 4 weeks of artificial tears in the temporal and inferior bulbar specimens. After 6 weeks of cyclosporine emulsion, goblet cell density was significantly greater than baseline and artificial tears in the inferior bulbar conjunctiva (P < 0.01). After 12 weeks of cyclosporine emulsion, goblet cell density was significantly greater than baseline and artificial tears in both temporal and inferior bulbar sites (P < 0.01). The number of TGF-beta2-positive goblet cells was also noted to increase after 6 and 12 weeks of cyclosporine therapy (P < 0.001)., Conclusions: Cyclosporine emulsion, but not artificial tears, increases goblet cell density and production of the immunoregulatory factor TGF-beta2 in the bulbar conjunctiva in patients with dry eye.

Purpose: To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation., Methods: The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at -196 degrees C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at -196 degrees C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture., Results: HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% +/- 0.8% and 79.8% +/- 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 +/- 1.35%, 2.31 +/- 2.23%, and 1.94 +/- 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% +/- 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% +/- 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% +/- 1.9% in cells with 20% FBS and glycerol and 13.5% +/- 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined., Conclusions: The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.

Purpose: To evaluate the effects of desiccating ocular surface stress on levels of inflammatory cytokines in the corneal epithelium, conjunctiva, and tear fluid of BALB/c and C57BL/6 mice., Methods: Experimental dry eye (EDE) was created in BALB/c and C57BL/6 mice by cholinergic blockade and exposure to a desiccating environment. Real-time polymerase chain reaction was performed to measure levels of cytokine transcripts. A multiplex immunobead assay was used to measure concentrations of these cytokines in tears., Results: Experimental dryness significantly increased the expression of interleukin (IL)-1alpha, IL-6, and tumor necrosis factor (TNF)-alpha transcripts in the corneal epithelium and conjunctiva of C57BL/6 mice. Strain-specific changes in tear cytokine profiles were observed. C57BL/6 mice had significantly greater tear concentrations of IL-1alpha and TNF-alpha and the Th-1 cytokines IL-2, IL-12, and interferon-gamma in response to desiccating stress than BALB/c mice. The Th-2 cytokines IL-4 and IL-10 were significantly greater in BALB/c tears., Conclusions: This study indicates that desiccating stress increases levels of certain cytokines in the corneal epithelium and conjunctiva in a strain-dependent fashion and that C57BL/6 mice had greater levels of Th-1 cytokines in their tears, whereas BALB/c mice had a greater increase in Th-2 cytokines.

Purpose: Mucins are highly glycosylated proteins that act as lubricants, protectants, and mediators of signal transduction. The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family members initiate mucin-type O-glycosylation. Because O-glycosylation provides mucins with the viscoelastic properties required for proper mucin function, ppGaNTase expression is vital to the maintenance of healthy epithelial surfaces including the ocular surface. Differences of expression of ppGaNTase isoforms might be factors influencing susceptibility to dry eye disease. Therefore, we determined the expression of the ppGaNTase isoforms in normal human ocular surface tissues and the conjunctival epithelium from patients with aqueous-deficient dry eye., Methods: Expression of ppGaNTase isoforms was quantitated by real-time reverse transcriptase polymerase chain rection (RT-PCR)., Results: Conjunctiva and cornea expressed multiple ppGaNTase isoforms, with isoforms T12 and T4 being the most strongly expressed in conjunctiva and T12 and T3 in the cornea. In contrast, lacrimal gland expressed fewer isoforms and had lower total ppGaNTase expression. Brush cytology was found to be superior to impression cytology for harvesting conjunctival epithelium in terms of ease of use, safety, and reproducibility of results. Similar to whole conjunctiva, the strongest isoforms in conjunctival epithelial cells were T12 and T4, followed by T3, T1, T5, and T2. No significant differences of ppGaNTase expression were found between the conjunctival epithelium of dry eye and normal control groups., Conclusion: Human ocular surface tissues express multiple ppGaNTase isoforms, suggesting a requirement for glycosylating diverse mucin-type substrates. However, there is no evidence to date to suggest that differences of ppGaNTase expression levels might contribute to susceptibility to dry eye disease.

Purpose: Tissue remodeling, known to accompany exacerbation of bronchial asthma (BA) and characterized by thickening of reticular basement membrane, increased fibrosis, and angiogenesis, is believed to be an important mechanism in giant papillary formation in vernal keratoconjunctivitis (VKC). This study was conducted to confirm the difference of tissue remodeling between BA and VKC and to determine the most relevant factor for VKC exacerbation., Methods: Histopathologic analysis of conjunctival specimens from 6 patients with VKC and 4 normal controls was performed. Immunohistochemistry tests for collagen types 1, 3, and 5, fibronectin, laminin (a marker of the basement membrane of the epithelium or vessels), fibroblast (prolyl 4-hydroxylase), and alpha-smooth muscle actin (alpha-SMA) were performed. Morphometric analysis evaluated immunoreactive areas for collagens and fibronectin and the number of cell nuclei in VKC papillae and normal control specimens., Results: In histopathologic sections of VKC papillae, mononuclear cells were mainly observed in the central region and granulocytes preferentially in the peripheral stroma. Immunohistochemistry showed increased vascularity and existence of fibroblasts and alpha-SMA-positive cells in VKC papillae. Morphometric analysis showed that the number of cell nuclei in VKC papillae was 7.3 times higher versus in normal controls (P < 0.05). Furthermore, the immunoreactive area for fibronectin was 4.5 times larger versus in normal controls (P < 0.05), although collagens were not significantly different between the 2 groups., Conclusion: Giant papillary formation in VKC is thought to occur in relation to tissue remodeling. However, the most relevant factor for the exacerbation of VKC seems to be inflammation rather than fibrosis.

Purpose: To determine how ocular surface epithelium adjusts to an increase in cell replication after treatment with the opioid antagonist naltrexone (NTX)., Methods: Adult male rats were given twice daily injections of 30 mg/kg NTX or vehicle for 7 days. Outcomes of NTX administration included DNA synthesis (monitored with BrdU), mitosis (assayed using colchicine), number of cell layers and cell diameter, apoptosis and necrosis, and packing density for the peripheral corneal epithelium, limbus, and conjunctiva. Also, transit time from basal to surface epithelial layers in the peripheral cornea was assessed with [H]thymidine as a marker., Results: DNA synthesis and mitosis in the basal layer of the peripheral corneal epithelium of NTX-treated rats were increased 69% and 85%, respectively, from control levels; no changes in either parameter were recorded in the limbal or conjunctival epithelium (stem cell region). Epithelial thicknesses in the NTX group were increased by 8% to 38% from control subjects, without more cell layers. Packing density in NTX-treated rats was increased from control values by 26% in the basal layer of the limbus and by 12% to 28% in the suprabasal layers of the corneal epithelium, limbus, and conjunctiva. Epithelial cell diameters from corneas of NTX-exposed rats were subnormal in the basal and suprabasal cells of the limbus and conjunctiva. Apoptosis and necrosis were negligible in the epithelium of NTX-treated and control rats. Transit times of peripheral corneal epithelial cells of animals in the NTX group were shortened by 63% from control levels., Conclusions: These data show that a 1-week treatment with NTX does not induce proliferative pathology or toxicity in ocular surface epithelium, has a minimal effect on stem cell proliferation, and accelerates normal homeostatic processes. Topical application of NTX for stimulation of corneal epithelial wound healing results in no adverse sequelae, thereby supporting the therapeutic role for this drug in the treatment of ocular surface abnormalities.

Purpose: To determine the pattern and distribution of mononuclear cells, adhesion, and co-stimulatory molecules in the conjunctiva of patients with Mooren ulcer., Methods: Conjunctival biopsy specimens were obtained from 6 patients with Mooren ulcer and 6 healthy individuals. Immunohistochemistry was performed on frozen sections of the cryopreserved human conjunctivas using monoclonal antibodies directed against CD1alpha, CD3, CD4, CD8, CD20, CD25, CD57, and CD68 cells; the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), very late activation-4 (VLA-4), ICAM-1, and LFA-1; and the co-stimulatory molecules CD28, B7-1, B7-2, and CTLA-4., Results: Differences in expression on the conjunctival epithelium from patients with Mooren ulcer and normal subjects were noted only for VCAM-1, VLA-4, ICAM-1, and LFA-1. The ratio of CD4+/CD8+ cells in Mooren ulcer specimens was significantly higher (3.5-fold). However, in the substantia propria, Mooren ulcer specimens revealed significantly increased numbers of CD1alpha+, CD3+, CD4+, CD20+, CD28+, B7-1+, B7-2+, and CD68+ cells. The ratios of CD4+/CD8+ cells and B7-2+/antigen-presenting cells in Mooren ulcer specimens were significantly higher (5-fold). All tested adhesion molecules showed significant up-regulation in the patients' conjunctivas. Mooren ulcer vascular endothelial cells prominently expressed E-selectin, VCAM-1, VLA-4, and ICAM-1 compared with normal conjunctiva., Conclusion: The simultaneous presence of multiple types of inflammatory cells, adhesion, and co-stimulatory molecules in Mooren ulcer conjunctiva suggests that their interaction may contribute to a sustained immune activation as at least part of the pathogenic mechanism of this disorder.

Purpose: To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture., Methods: Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody., Results: As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4., Conclusions: The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Purpose: This study was designed to determine whether long-term tolerant contact lens (CL) wear causes changes in the expression of mucin mRNA by the conjunctival epithelium and mucin protein content in tears and to determine whether specific mucins adhere to contact lenses., Methods: Twenty long-term (> or = 5 years ) and tolerant CL wearers (2 with hard and 18 with soft contact lenses) were compared with 23 non-CL wearers. One hour after CL removal, tear fluid was collected after instillation of 60 microL of sterile water onto the ocular surface, and protein concentration was determined. Impression cytology was performed on the bulbar temporal region of conjunctiva to collect cells for RNA isolation. Real-time polymerase chain reaction was performed using TaqMan primer and probes for MUC1, 4, 5AC, and 16. ELISA was performed on the collected tears to detect MUC5AC and the mucin carbohydrate epitope H185. For the analysis of adherent mucins on CL, discarded daily-wear contact lenses were collected, rinsed, and incubated overnight at 4 degrees C in mucin isolation buffer. Immunoblot analysis of adherent mucins was performed to detect MUC1, 4, 5AC, 16, and H185., Results: No significant changes in the levels of mucin mRNA from impression cytology samples were detected when comparing CL and non-CL wearers. The amount of total protein in tears collected from CL wearers (39.9 +/- 27.2 microg) was significantly less than that from non-CL wearers (95.1 +/- 73.8 microg, P = 0.001). The level of MUC5AC mucin and the H185 epitope in tears per unit protein in CL wearers was not significantly different from non-CL wearers. Low levels of membrane-associated mucins, the secreted mucin MUC5AC, and the carbohydrate epitope, H185, were detected in protein extracts from discarded CLs. Compared with MUC1, 4, and 5AC, there was less MUC16 adherent to the CLs., Conclusion: Neither mucin mRNA expression by conjunctival epithelia nor mucin content per unit protein in tears was altered by long-term tolerant CL wear; however, the amount of protein in the tears was significantly less. Shed membrane-associated mucins and the goblet cell mucins adhere to CLs.

Purpose: To examine the expression of HLA-DR, a marker of inflammation, in the early stages of dry eye disease and to locate the appearance of this marker on specific areas of the bulbar conjunctiva., Methods: Dry eye patients were identified and their condition classified as mild (n = 16) or moderate (n = 16) based on Schirmer testing, vital staining, tear break-up time, and symptom questionnaire scores. Brush cytology was used to collect epithelial cells from the nasal, temporal, and superior conjunctivae of patients and age-matched controls. HLA-DR positive cells were detected by immunohistochemical staining and quantified., Results: Patients with moderate dry eye had the highest rate of conjunctival HLA-DR-positive cells, with significantly higher rates than controls regardless of which region of the conjunctiva was sampled (P < 0.01). The mild dry eye group had similar rates of HLA-DR-positive cells in the superior conjunctival region compared with controls. However, in the nasal and temporal regions, they displayed a significantly higher rate of HLA-DR-positive cells than controls (P < 0.01) and the nasal region showed a significant difference (P < 0.01) when compared with the temporal one. Some of these mild dry eyes had no vital staining., Conclusions: The HLA-DR expression pattern in mild and moderate dry eyes appears to reflect disease progression. Overexpression of HLA-DR in mild dry eyes showing no vital staining suggests that inflammation may be a primary cause of ocular surface damage. These data support the use of immunomodulatory drugs in the treatment of dry eye disease.

Purpose: To determine whether conjunctival lymphoid follicles are preferential sites for uptake of foreign material in the preocular tear film., Methods: Iron oxide suspension was applied to the eyes of New Zealand white rabbits for selected times, and the conjunctiva was examined histochemically for the presence of iron., Results: Iron was observed by histochemical staining within conjunctival follicle-associated epithelium at 1 hour and deep within the follicles at 4 hours after exposure. Iron was not seen within nonfollicular conjunctival epithelium or underlying substantia propria at any time after iron oxide application., Conclusions: Iron oxide in the preocular tear film is taken up preferentially by conjunctival lymphoid tissue, supporting the hypothesis that mammalian conjunctival lymphoid follicles may participate in the acquired immune response to pathogens in the preocular tear film.

Purpose: Rebamipide increases the amount of mucin-like substances in the stomach. We aimed to determine the effects of rebamipide on the amount of mucin-like substances in the conjunctiva and cornea of N-acetylcysteine-treated eyes. Furthermore, we attempted to evaluate the effects of rebamipide on the wound healing of N-acetylcysteine-treated eyes., Methods: The model was created by instilling 10% N-acetylcysteine solutions into rabbit eyes. Rebamipide was then applied on the day following the completion of N-acetylcysteine treatment. The amount of mucin-like substances on the conjunctiva and cornea was measured using the Alcian-blue binding method. The degree of damage was evaluated using scores based on the areas and densities of the cornea and conjunctival after staining using a rose Bengal solution under blind conditions., Results: Rebamipide increased the level of mucin-like substances on the conjunctiva of N-acetylcysteine-treated eyes when instilled at concentrations of 0.3% or higher, and 1% rebamipide increased the amount of mucin-like substances covering the cornea. Moreover, 1% rebamipide improved the rose Bengal scores of the cornea and conjunctiva in N-acetylcysteine-treated eyes., Conclusions: Rebamipide increased mucin-like substances on the cornea and conjunctiva of N-acetylcysteine-treated eyes. In accordance with the mucin-increasing effects, rebamipide improved the rose Bengal scores for the cornea and conjunctiva of N-acetylcysteine-treated eyes. However, the relevance of these findings to dry eyes is unclear because it is not known whether the change in mucus expression in the N-acetylcysteine model is similar to what occurs in aqueous tear deficiency. Consequently, it may be worth trying on an animal model of keratoconjunctivitis sicca.

Purpose: The p63 gene has been identified as a marker of epithelial stem cells. Because pterygium may arise through an expansion of the proliferative capacity of the conjunctiva, we sought to document the expression of p63 in normal conjunctiva and pterygium specimens., Methods: Immunostaining for p63 expression was performed on 10 pairs of pterygium and normal conjunctiva using a monoclonal antibody directed against human p63., Results: Immunopositive reactions were seen in all samples. Levels of p63-positive cells were not statistically different between pterygium and normal conjunctivae (P = 0.7)., Conclusion: These results strongly support previous studies that indicate that pterygium arises as a result of incorrect control of cellular apoptosis rather than from an increase in proliferative capacity.

Purpose: To prove that noninvasive methods of obtaining conjunctival tissue, such as conjunctival impression cytology (CIC), could be valid alternatives that are simpler, faster, and more convenient for patients than biopsy to analyze mRNA levels of mucin genes., Methods: Using the semiquantitative reverse-transcriptase polymerase chain reaction, we studied the presence of the mucin genes described on the ocular surface thus far and attempted to detect the presence of MUC7 in CIC samples from 10 healthy donors., Results: Conjunctival cells recovered by CIC expressed all the genes studied. There were no statistically significant differences between male and female subjects, and there was a significant correlation between the two eyes of the same donor only in the expression of MUC7., Conclusion: CIC is a valid, noninvasive technique to detect the mRNAs of ocular genes in healthy individuals. MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes could be all detected in each CIC sample. This technique may be a useful tool to study the expression of some genes in ocular diseases.

Purpose: Recent research indicates that epithelial cells of the ocular surface can contribute to the allergic reaction by the release of inflammatory and/or chemotactic mediators. In this study, the role of two inflammatory mediators, previously identified in the tear film of ocular allergy subjects, TNF-alpha and IFN-gamma, were evaluated for their effect on the release of two chemotactic mediators, IL-8 and RANTES, from cultured human conjunctival epithelial cells., Methods: Human conjunctival epithelial cells (primary cells or HC0597 cell line) were grown to confluence and stimulated with various concentrations of TNF-alpha, IFN-gamma, or a combination of both. Supernatants were collected at 6, 24, and 48 hours and stored frozen for subsequent ELISA analyses of RANTES and IL-8., Results: RANTES and IL-8 release from HC0597 cells was stimulated in a dose- and time-dependent manner following treatment with TNF-alpha. However, only RANTES release was modulated by IFN-gamma treatment. Treatment of HC0597 cells with both TNF-alpha and IFN-gamma resulted in a synergistic increase in the release of RANTES. This synergistic effect was confirmed using primary cultures of human conjunctival epithelial cells., Conclusions: Stimulation of conjunctival epithelium with proinflammatory mediators, TNF-alpha and/or IFN-gamma, generated the release of the chemotactic factors IL-8 and RANTES, which could act to prolong inflammation. These two chemokines may prolong inflammation by recruiting eosinophils to the ocular surface. This is the first study to compare chemokine release in a cell line and primary cells; similar chemokine release after mediator stimulation was demonstrated, indicating that the two cell types are phenotypically similar.