Human Conjunctiva Research Articles

Calcium-sensing receptor (CaSR) modulates ocular surface chloride transport and its inhibition promotes ocular surface hydration

PurposeOcular surface hydration is critical for eye health and its impairment can lead to dry eye disease. Extracellular calcium-sensing receptor (CaSR) is regulator of ion transport in epithelial cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. CFTR is also a major ion channel in ocular surface epithelia, however the roles of CaSR in ocular surface are not well studied. This study aims to investigate expression and functional roles of CaSR in ocular surface. MethodsCaSR immunostaining was performed in mouse and human cornea and conjunctiva. Ocular surface potential difference (OSPD) and tear fluid volume measurements were performed in mice with topically applied cinacalcet (CaSR activator) and NPS-2143 (CaSR inhibitor). ResultsCaSR is expressed in corneal and conjunctival epithelia of mice and humans. Topically administered CaSR activator cinacalcet inhibits cAMP agonist forskolin-induced Cl− secretion and CFTR activity up to 90 %. CaSR inhibitor NPS-2143 stimulates CFTR-mediated Cl− secretion in mouse ocular surface, after which cAMP agonist forskolin had minimal additional secretory effects. Single dose NPS-2143 treatment (as an eye drop) increases tear fluid volume in mice by ∼60 % compared to vehicle treatment. NPS-2143 effect on tear volume lasts at least 8 h after single dose. ConclusionsCaSR is a key regulator of ocular surface ion transport and CaSR inhibition promotes Cl− and tear secretion in the ocular surface. If they are found to be effective in in dry eye models, CaSR inhibitors (currently in clinical development) can potentially be repurposed as novel prosecretory treatments for dry eye disease.

Molecular detection of lacrimal apparatus and ocular surface - related ABC transporter genes

The ocular system is in constant interaction with the environment and with numerous pathogens. The ATP-binding cassette (ABC) transporters represent one of the largest groups among the transmembrane proteins. Their relevance has been demonstrated for their defense function against biotic and abiotic stress factors, for metabolic processes in tumors and for their importance in the development of resistance to drugs. The aim of this study was to analyze which ABC transporters are expressed at the ocular surface and in the human lacrimal apparatus. Using RT-PCR, all ABC transporters known to date in humans were examined in tissue samples from human cornea, conjunctiva, meibomian glands and lacrimal glands. The RT-PCR analyses revealed the presence of all ABC transporters in the samples examined, although the results for some of the 48 transporters known in human and analyzed were different in the various tissues. The present results provide information on the expression of ABC transporters at the mRNA level on the ocular surface and in the lacrimal system. Their detection forms the basis for follow-up studies at the protein level, which will provide more information about their physiological significance at the ocular surface and in the lacrimal system and which may explain pathological effects such as drug resistance.

Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer.

Transthyretin amyloidosis (ATTR) is a significant cause of cardiomyopathy and other morbidities in the elderly and Black Americans. ATTR can be treated with new disease-modifying therapies, but large shortfalls exist in its diagnosis. The objective of this study was to test whether TTR amyloid can be detected and imaged in the conjunctiva using a novel small-molecule fluorescent ocular tracer, with the implication that ATTR might be diagnosable by a simple eye examination. Three approaches were used in this study. First, AMDX-9101 was incubated with in vitro aggregated TTR protein, and changes in its excitation and emission spectra were quantified. Second, a cadaver eye from a patient with familial amyloid polyneuropathy type II TTR mutation and a vitrectomy sample from an hATTR patient were incubated with AMDX-9101 and counterstained with Congo Red and antibodies to TTR to determine whether AMDX-9101 labels disease-related TTR amyloid deposits in human conjunctiva and eye. Last, imaging of in vitro aggregated TTR amyloid labeled with AMDX-9101 was tested in a porcine ex vivo model, using a widely available clinical ophthalmic imaging device. AMDX-9101 hyper-fluoresced in the presence of TTR amyloid in vitro, labeled TTR amyloid deposits in postmortem human conjunctiva and other ocular tissues and could be detected under the conjunctiva of a porcine eye using commercially available ophthalmic imaging equipment. AMDX-9101 enabled detection of TTR amyloid in the conjunctiva, and the fluorescent binding signal can be visualized using commercially available ophthalmic imaging equipment. AMDX-9101 detection of TTR amyloid may provide a potential new and noninvasive test for ATTR that could lead to earlier ATTR diagnosis, as well as facilitate development of new therapeutics.

The existence of senescent cells in conjunctival epithelium from elderly individuals.

The ocular surface microenvironment changes with aging. However, it remains unclear if cellular senescence influences the ocular surface. We investigated the presence of p16INK4a-expressing senescent cells in healthy human conjunctiva. Clinical and experimental. Healthy conjunctival tissue samples were obtained from middle-aged and elderly subjects. RT-qPCR was performed to assess the expression of senescence markers CDKN2A (p16INK4a) and CDKN1A (p21CIP1/WAF1) and immunostaining was performed to examine the expression of the senescence marker p16INK4a, stem cell markers Ki67 and p63, tight-junction marker ZO-1. Our study involved 19 conjunctival tissue samples (10 elderly and 9 middle-aged), mean age [elderly: 75.8 ± 3.7 years (72-81), middle-aged: 52.7 ± 7 years (38-59)], sex (elderly: 3 men, 7 women; middle-aged: 3 men, 6 women). The expression of p16INK4a was significantly increased at the RNA level in the elderly compared to middle-aged (p < 0.05). Positivity rate of p16INK4a was significantly elevated in the elderly (15.0 ± 7.8%) compared to middle-aged (0.2 ± 0.6%) (p < 0.05). Positivity rate of Ki67and p63 was significantly reduced in the elderly (1.7 ± 1.7% and 16.5 ± 9.5%) compared to middle-aged (3.9 ± 1.8% and 24.7 ± 5.7%) (p < 0.05). ZO-1 expression was reduced in tissue samples showing p16INK4a-positivity but retained in tissue samples in which p16INK4a was undetectable. Senescent cells accumulate with age in the conjunctival epithelium, accompanied by a decrease in Ki67, p63 and ZO-1 expressing cells.

Human conjunctiva organoids to study ocular surface homeostasis and disease

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Transcriptional profilling to identify the inhibitor effect of trehalose as an alternative natural therapeutic agent on pterygium cells

Aims/Purpose: The aim of this study to investigate transcriptome analysis of the inhibitory effects of trehalose on changing expression of regulator genes in pterygium development.Methods: Human primary pterygium cells and normal conjunctiva cells were isolated from the pterygium and conjunctiva tissues of six individuals, three of whom were men and three of which were females. The MTT test was used to investigate cell proliferation in pterygium treated with different concentrations dosage of trehalose. Differential mRNA expression in pterygium cells following indicative treatment was measured using an mRNA microarray and evaluated using GO and KEGG pathways. In addition, we employed QRT‐PCR to confirm the expression levels of genes linked to pterygium pathogenesis.Results: We have found that pterygium cells are more sensitive to trehalose treatment than conjunctival cell. In total, 419 probe were differentially expressed pterygium cells treated with trehalose compared with paired non‐treated pterygium cells. Gene‐Enrichment and Functional Annotation analysis indicated that differentially expressed RNAs were associated with cellular process including especially fibroblastic cell migration, sterol biosynthetic process (p &lt; 0.00034), extracellular matrix recomposition (p &lt; 0.000740288), cell proliferation (p &lt; 0.004391252), steroid biosynthetic process (p &lt; 0.00029971). Composed of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein–protein interaction (PPI), and gene set enrichment analysis (GSEA) analysis, bioinformatic analyses showed that four hub genes were upregulated (MSMO, RANBP3L, DHCR7, CCL2) and five were downregulated (NR4A1, TENASTN C, CXCL8, CXCL12, ADAMTS12). Additionally, QRT‐PCR is used to confirm the expression of particular genes. Consistent with these findings, the migration capability of pterygium cells decreased after treatment of trehalose for 24 h.Conclusions: We consider that this study provides a scientific basis for elucidating the effect of trehalose on the pathogenesis of pterygium and exhibited that trehalose affects cellular processes that have essential roles in the progression and development of pterygium.

Single-cell landscape reveals the epithelial cell-centric pro-inflammatory immune microenvironment in dry eye development

Dry eye disease (DED) is a prevalent chronic eye disease characterized by an aberrant inflammatory response in ocular surface mucosa. The immunological alterations underlying DED remain largely unknown. In this study, we employed single-cell transcriptome sequencing of conjunctival tissue from environment-induced DED mice to investigate multicellular ecosystem and functional changes at different DED stages. Our results revealed an epithelial subtype with fibroblastic characteristics and pro-inflammatory effects emerging in the acute phase of DED. We also found that T helper (Th)1, Th17, and regulatory T cells (Treg) were the dominant clusters of differentiation (CD)4+ T-cell types involved in regulating immune responses and identified three distinct macrophage subtypes, with the CD72+CD11c+ subtype enhancing chronic inflammation. Furthermore, bulk transcriptome analysis of video display terminal-induced DED consistently suggested the presence of the pro-inflammatory epithelial subtype in human conjunctiva. Our findings have uncovered a DED-associated pro-inflammatory microenvironment in the conjunctiva, centered around epithelial cells, involving interactions with macrophages and CD4+ T cells, which deepens our understanding of ocular surface mucosal immune responses during DED progression.

Ocular Pharmacology and Toxicology of TRPV1 Antagonist SAF312 (Libvatrep).

To evaluate the pharmacology and toxicology of SAF312, a transient receptor potential vanilloid 1 (TRPV1) antagonist. TRPV1 expression in human ocular tissues was evaluated with immunohistochemistry. Inhibition of calcium influx in Chinese hamster ovary (CHO) cells expressing human TRPV1 (hTRPV1) and selectivity of SAF312 were assessed by a fluorescent imaging plate reader assay. Ocular tissue and plasma pharmacokinetics (PK) were assessed following a single topical ocular dose of SAF312 (0.5%, 1.0%, 1.5%, 2.5%) in rabbits. Safety and tolerability of SAF312 were evaluated in rabbits and dogs. Effects of SAF312 on corneal wound healing after photorefractive keratectomy (PRK) surgery were assessed in rabbits. TRPV1 expression was noted in human cornea and conjunctiva. SAF312 inhibited calcium influx in CHO-hTRPV1 cells induced by pH 5.5 (2-[N-morpholino] ethanesulfonic acid), N-arachidonoylethanolamine, capsaicin, and N-arachidonoyl dopamine, with IC50 values of 5, 10, 12, and 27 nM, respectively, and inhibition appeared noncompetitive. SAF312 demonstrated high selectivity for TRPV1 (>149-fold) over other TRP channels. PK analysis showed highest concentrations of SAF312 in cornea and conjunctiva. SAF312 was found to be safe and well tolerated in rabbits and dogs up to the highest feasible concentration of 2.5%. No delay in wound healing after PRK was observed. SAF312 is a potent, selective, and noncompetitive antagonist of hTRPV1 with an acceptable preclinical safety profile for use in future clinical trials. SAF312, which was safe and well tolerated without causing delay in wound healing after PRK in rabbits, may be a potential therapeutic agent for ocular surface pain.

TGF-β Isoforms Affect the Planar and Subepithelial Fibrogenesis of Human Conjunctival Fibroblasts in Different Manners.

Three highly homologous isoforms of TGF-β, TGF-β-1~3, are involved in the regulation of various pathophysiological conditions such as wound healing processes in different manners, despite the fact that they bind to the same receptors during their activation. The purpose of the current investigation was to elucidate the contributions of TGF-β-1 ~3 to the pathology associated with conjunctiva. For this purpose, the biological effects of these TGF-β isoforms on the structural and functional properties of two-dimensional (2D) and three-dimensional (3D) cultured human conjunctival fibroblasts (HconF) were subjected to the following analyses: 1) transendothelial electrical resistance (TEER), a Seahorse cellular metabolic measurement (2D), size and stiffness measurements of the 3D HTM spheroids, and the qPCR gene expression analyses of extracellular matrix (ECM) components (2D and 3D). The TGF-β isoforms caused different effects on the proliferation of the HconF cell monolayer evaluated by TEER measurements. The differences included a significant increase in the presence of 5 ng/mL TGF-β-1 and -2 and a substantial decrease in the presence of 5 ng/mL TGF-β-3, although there were no significant differences in the response to the TGF-β isoforms for cellular metabolism among the three groups. Similar to planar proliferation, the TGF-β isoforms also induced diverse effects toward the mechanical aspects of 3D HconF spheroids, where TGF-β-1 increased stiffness, TGF-β-2 caused no significant effects, and TGF-β-3 caused the downsizing of the spheroids and stiffness enhancement. The mRNA expression of the ECMs were also modulated in diverse manners by the TGF-β isoforms as well as the culture conditions for the 2D vs. 3D isoforms. Many of these TGF-β-3 inducible effects were markedly different from those caused by TGF-β1 and TGF-β-2. The findings presented herein suggest that the three TGF-β isoforms induce diverse and distinctly different effects on cellular properties and the expressions of ECM molecules in HconF and that these changes are independent of cellular metabolism, thereby inducing different effects on the epithelial and subepithelial proliferation of human conjunctiva.

Effect of immobilized hyaluronidase and subtilisin enzymes on the ultrastructure of human conjunctival epithelial cells

The use of enzyme preparations is a traditional trend in various fields of medicine. The usage of immobilized enzymes hyaluronidase (IG) and subtilisin (IS) seems to be very promising for the treatment of damage to the ocular surface. The aim of the study was to establish the nature of changes in the ultrastructural organization of human conjunctival epithelium under the influence of immobilized hyaluronidase and subtilisin in vitro. Material and methods. In the experiment, a culture of normal cells of the human conjunctiva Chanqconjunctiva, clone 1–5 C-4, was seeded in 96well plates in the amount of 2 × 104 cells/well, after 24 h the medium was removed, Eagle’s medium MEM and fetal calf serum were added and cells were cultured for another 48 h. 5 experimental groups were formed: group 1 – without drugs (except for the composition of the incubation scheme), groups 2 and 3 – with IS at a concentration of 37 and 150 U/ml, respectively, groups 4 and 5 – with IG at a concentration of 37 and 150 U/ml, respectively. After preparation of cell preparations under a JEM 1400 electron microscope (Japan), ultrathin sections of 70–100 nm were studied at ×1000 magnification. Results. The article presents the results of electron microscopy of a culture of normal cells of the human conjunctiva in 5 experimental groups of cells with a description of changes in cytoarchitectonics under the influence of IG and IS. Conclusions. The introduction of immobilized enzymes into human conjunctival cell culture at a low dose (37 U/ml) affects the organization of cells without cytotoxic effects, while increasing the dose directly correlates with the occurrence of a cytotoxic effect.

Expression of GPR-68 in Human Corneal and Conjunctival Epithelium. Possible indicator and mediator of attrition associated inflammation at the ocular surface

Attrition and osmotic stress have been identified as major forces in the pathophysiology of dry eye. Impaired tolerance to mechano-transduction in the presence of insufficient lubrication has been associated with disturbances of ocular surface homeostasis and encouragement of inflammatory reactions, challenging the usual regulatory coping mechanisms. In spite of the probable link between enhanced attrition and secondary inflammation, the key mediators driving the vicious cycle of severe dry eye disease have not yet been identified. The goal of this study was therefore to investigate human corneal and conjunctival epithelium for the presence of the G protein-coupled receptor GPR-68. This protein had most recently been shown to be not only chemically activated but also mechanically, possibly through attrition. De-identified sections of human cornea and conjunctiva were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemical methods. Results Specific staining for G-protein-coupledreceptor 68 (GPR68) was observed in all samples of the cornea throughout the epithelial layers of the corneal epithelium, most prominently in the area of the wing cells and the basement membrane. Even in the conjunctiva, specific staining for GPR-68 was found. The detection of G protein-coupled receptor GPR-68 in human corneal and conjunctival epithelium raises the question of its function and purpose. The mechanical activation of GPR68 in situations with enhanced friction and attrition could modify various cellular functions and possibly jeopardize normal inflammatory homeostasis at the ocular surface. Accordingly, decreased lubrication in dry eye disease could result in activation of GPR-68. This could lead to secondary inflammation, initially in the epithelium and surrounding stroma. Continuous mechanical stress could result in chronic inflammation, also reaching deeper structures of the cornea, possibly making GPR-68 an important actor in the vicious cycle of dry eye disease. G protein-coupled receptor GPR-68, sensitive to flow and mechanic stimulation, is present in the human corneal epithelium and conjunctiva. Decreased lubrication and increased attrition, accompanied by sensations typical for dry eye, might lead to local inflammation. It is possible that subtle signs of conjunctival, and later corneal, surface damage in the context of these sensations could be a better indicator of the need for and success of therapy than the clinical signs of dry eye disease alone, at least in the early stages of the disease. Inhibition of G protein-coupled receptor GPR-68 could represent a new strategy in the treatment of dry eye disease.

Conjunctiva Resident γδ T Cells Expressed High Level of IL-17A and Promoted the Severity of Dry Eye.

Conjunctival inflammation promotes ocular surface disorders in dry eye disease (DED). Here we identified γδ T cells as the predominant source of IL-17A in the murine conjunctiva and assessed their contribution to the pathogenesis of DED. We enrolled 22 patients with DED, and analyzed the proportion of γδ T cells in the conjunctival epithelial samples by flow cytometry. Adult C57Bl/6 wild-type and TCRδ-/- mice were used to induce DED models to investigate the role of γδ T cells. The characteristics of immune cell infiltration and the expression of immune-related cytokines or markers in mouse conjunctiva were analyzed by flow cytometry, Western blot, and quantitative polymerase chain reaction. The proportion of γδ T cells in the human DED conjunctiva is significantly higher in patients with severe corneal epithelial defects than in mild ones, which is consistently observed in the murine DED model. Further, a high level of IL-17A but not IFN-γ is detected in the conjunctiva of mice. The increased murine IL-17A-producing cells on the conjunctiva are identified as γδ T cells predominantly and Th17 cells to a lesser extent. Ablation of γδ T cells by antibody depletion or genetic deletion of TCRδ alleviates ocular surface damage in the murine DED model. Our studies evaluate human and experimental murine DED for evidence of γδ T-cell-mediated inflammation and highlight a potential therapeutic synergy by targeting IL-17 and γδ T cells in DED treatment.

Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators

Many conjunctival inflammatory diseases differ between the sexes and altered conjunctival goblet cells (CGCs) response is often involved. Inflammation is initiated by the release of pro-inflammatory mediators and terminated by the biosynthesis of specialized pro-resolution mediators (SPMs). Herein, we determined the sex-based difference in the responses of CGCs to inflammatory stimuli or pro-resolving lipid SPMs and their interaction with sex hormones. GCs were cultured from pieces of human conjunctiva in RPMI media. CGCs were transferred 24 h before the start of experiments to phenol red-free and FBS-free media to minimize exogenous hormones. RT-PCR, immunofluorescence microscopy (IF), and Western Blot (WB) were performed to determine the presence of sex hormone receptors. Cellular response to pro-inflammatory stimuli or SPMs was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) using fura 2/AM microscopy. Use of RT-PCR demonstrated estrogen receptor (ER) α in 4/5 males and 3/3 females; ERβ in 2/4 males and 2/3 females; and androgen receptors (AR) in 3/3 male and 3/3 female CGCs. Positive immunoreactivity by IF and protein expression by WB was detected using antibodies for the ERα and ERβ in 3/3 males and 3/3 females, while AR were only present in males. Significantly different Ca2+ responses between sexes were found with carbachol only at 10–3 M, but not with histamine or leukotriene (LT) B4 at any concentration used. Incubation with dihydrotestosterone (DHT), estrone (E1), or estradiol (E2) at 10–7 M for 30 min significantly inhibited the LTB4-stimulated [Ca2+]i increase in male and female CGCs. Incubation with DHT, E1, and E2 overnight significantly inhibited the LTB4 response in females, while DHT and E2 significantly inhibited the LTB4 response in males. The SPM lipoxin A4 (LXA4) (10–9–10−8 M), but not the resolvins D1 or D2, induced an [Ca2+]i increase that was significantly higher in males compared to females. We conclude that male and female CGCs showed differences in the expression of sex hormone receptors. Treatment with sex hormones altered pro-inflammatory mediator LTB4-induced response. Males compared to females have a higher response to the ω-6-fatty acid derived SPM LXA4, indicating males may terminate inflammation in conjunctival goblet cells faster than females.

Benzalkonium Chloride, Even at Low Concentrations, Deteriorates Intracellular Metabolic Capacity in Human Conjunctival Fibroblasts.

The objective of this study was to clarify the effects of benzalkonium chloride (BAC) on two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells, which are in vitro models replicating the epithelial barrier and the stromal supportive functions of the human conjunctiva. The cultured HconF cells were subjected to the following analyses in the absence and presence of 10−5% or 10−4% concentrations of BAC; (1) the barrier function of the 2D HconF monolayers, as determined by trans-endothelial electrical resistance (TEER) and FITC dextran permeability, (2) real-time metabolic analysis using an extracellular Seahorse flux analyzer, (3) the size and stiffness of 3D HconF spheroids, and (4) the mRNA expression of genes that encode for extracellular matrix (ECM) molecules including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), ER stress related genes including the X-box binding protein-1 (XBP1), the spliced XBP1 (sXBP1) glucose regulator protein (GRP)78, GRP94, and the CCAAT/enhancer-binding protein homologous protein (CHOP), hypoxia inducible factor 1α (HIF1α), and Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α). In the presence of BAC, even at low concentrations at 10−5% or 10−4%, the maximal respiratory capacity, mitochondrial respiratory reserve, and glycolytic reserve of HconF cells were significantly decreased, although the barrier functions of 2D HconF monolayers, the physical properties of the 3D HconF spheroids, and the mRNA expression of the corresponding genes were not affected. The findings reported herein highlight the fact that BAC, even such low concentrations, may induce unfavorable adverse effects on the cellular metabolic capacity of the human conjunctiva.

Wnt/β-catenin signaling stimulates the self-renewal of conjunctival stem cells and promotes corneal conjunctivalization

Limbal stem cell deficiency causes conjunctivalization characterized by the covering of the corneal surface with conjunctival epithelium. However, the driving force for the encroachment of these conjunctival cells is unclear. Conjunctival stem cells are bipotent stem cells that can proliferate and differentiate into conjunctival epithelial cells and goblet cells to maintain regeneration of the conjunctival epithelium. Here, we show a robust proliferative response of conjunctival stem cells and upregulation of Wnt2b and Wnt3a gene expression in the conjunctivae of mice with induced limbal stem cell deficiency. Topical application of the Wnt/β-catenin signaling activator CHIR resulted in increased proliferation of ΔNp63α-positive stem cells in the basal layers of the bulbar and forniceal conjunctivae and enhanced invasion of conjunctival epithelial and goblet cells into the corneal surface. We also found that in cultures of stem cells isolated from the human conjunctiva, Wnt/β-catenin pathway activation improved the expansion of the ΔNp63α/ABCG2 double-positive cell population by promoting the proliferation and preventing the differentiation of these cells. These expanded stem cells formed a stratified epithelium containing goblet cells under airlift culture conditions. Our data reveal that Wnt/β-catenin signaling contributes to the pathological process of limbal stem cell deficiency by promoting the self-renewal of conjunctival stem cells and suggest that these cells are a driving force in corneal conjunctivalization.

Matrix Metalloproteinase-14 as an Instigator of Fibrosis in Human Pterygium and Its Pharmacological Intervention.

There exists a paucity of information on the pathogenesis of pterygium, a benign ocular tumor that scars the cornea and can lead to vision loss. The main recourse for pterygium is surgery; however, recurrence is observed. Matrix metalloproteinases (MMPs) are involved in the pathology of pterygium. The determination of the specific MMP involved among the 24 human enzymes has not been established due to challenges in MMP profiling. We used an affinity resin that binds specifically to the active forms of MMPs in the complex mixture of the cellular proteome. The proteomics analysis identified active MMP-14 and three related metalloproteinases, ADAM9, ADAM10, and ADAM17, in human pterygia. Inhibition of MMP-14 with the small-molecule inhibitor (R)-ND-336 was assessed in cell migration and collagen contraction assays. (R)-ND-336 attenuated human conjunctiva fibroblast migration and mitigated collagen contraction, both activities required for the formation of pterygium. (R)-ND-336 holds the promise of a therapeutic recourse for pterygium as an orphan disease.

Proteasomes in the Cells of the Ocular Surface in Health and Disease

PurposeLimbal stem cell deficiency (LSCD) is associated with corneal conjunctivalization that compromises corneal transparency and causes visual impairment. Previously, we found marked changes in proteasome population in corneas of rabbit with LSCD, and showed immunoproteasome (IPR) presence in central corneas during conjunctivalization compared to healthy rabbit cornea. In the present study, we further map the distribution of proteasomes population in the ocular surface.Methodsgene expression intensity obtained by microarray analysis of human healthy corneas and conjunctiva previously reported by the group of Dr. Wolosin (Turner H.C. et al 2007) was used to conduct this study. Constitutive proteasome (CPR) subunits (B5, B2, and B1) were compared to the inducible immunoproteasome (IPR) subunits (B5i, B2i, and B1i). The ratio between CPR and IPR subunits was calculated for cornea and for conjunctiva.ResultsThe comparison of the ratio B5/B5i, B2/B2i and B1/B1i between cornea and conjunctiva showed a significant high expression of CPR in corneas and a high expression of IPR in conjunctivae. In addition, PA28, the regulatory complex of IPR, and the major histocompatibility complex, class I A and B, were found significantly expressed in conjunctiva. Keratins K3/K12 were specifically expressed in cornea and keratins K4/K13 were specifically expressed in conjunctiva, which could be used to normalize the data analysis.ConclusionThe results indicated that conjunctivae contain high levels of IPR and that during conjunctivalization, corneas lose CPR to express IPR. The ration CPR/IPR could be used as an indication of conjunctivalization level in case of corneal injuries.

Tissue resident memory T cells inhabit the deep human conjunctiva

Mucosal linings of the body, including the conjunctiva, are enriched in tissue-resident memory T cells (TRMs) whose defining feature is their continual tissue protection that does not rely on migration to lymphoid organs to elicit immune responses. Hitherto, conjunctival TRMs have only been identified in the superficial epithelium. This work aims to develop a more complete understanding of the conjunctival immunological capacity by investigating the presence of TRMs within the deeper, more stable layers of the healthy human conjunctiva. Using immunofluorescence microscopy and antibodies against CD3, CD4, CD69 and HLA-DR on bulbar conjunctival biopsies obtained from 7 healthy adults (age range = 32–77 years; females = 4), we identified CD69+TRM subsets in all layers of the human conjunctiva: the superficial epithelium, the basal epithelium, the adenoid, and the fibrous layers. Interestingly, the adenoid layer showed significantly higher densities of both CD4 and CD8 TRMs when compared to the fibrous layer and conjunctival epithelia. Additionally, CD4 TRMs predominated significantly over CD8 TRMs in the adenoid layer. The abundance of deep conjunctival CD69+TRMs within the healthy human may suggest the presence of defence mechanisms capable of inducing long-term immunogenic memory. Understanding this spatial distribution of conjunctival CD69+TRMs is essential to improving mucosal vaccine design.

490 A high-fidelity globe and orbit surgical simulator for ophthalmologic surgical training

OBJECTIVES/GOALS: Many ophthalmologic procedures involve operating on or manipulating the globe and bony orbit. Creating an anatomically accurate globe and orbit is of interest to improve surgical education for trainees. The purpose of this study was to create a high-fidelity globe and orbit model using synthetic materials and utilizing 3D-printing techniques. METHODS/STUDY POPULATION: A deidentified computed tomography scan of the head and neck was digitally rendered and segmented using Mimics and 3-Matic (Materialise NV, Belgium) to create a digital model of the bony orbit. The model was 3D printed using a stereolithographic 3D-printer (Formlabs, Somerville, MA). The globe was created by soaking a large water bead made from a water absorbing polymer (YIQUDUO, China) for 24 hours. The water bead was then coated consecutively with three layers of silicone (Smooth-On, Macungie, PA). A standard sausage casing was hydrated and encased around the water bead, representing the conjunctiva. The globe was placed into the 3D-printed orbit. An incision was made in the sausage casing and the defect was sutured by one ophthalmologist. RESULTS/ANTICIPATED RESULTS: The bony orbital anatomy was accurately represented by stereolithographic printing. The size and feel of the artificial globe was similar to that of an in vivo human globe. The incised sausage casing covering the globe was able to be manipulated and sutured using a 8-0 suture in a microsurgical environment. The sausage casing had high-fidelity characteristics of an in vivo human eye conjunctiva. DISCUSSION/SIGNIFICANCE: This model can be used for teaching of conjunctival suturing for ophthalmologic trainees. By use of easily obtained materials for the globe, this model has the potential to standardize teaching methods of challenging techniques, and can reduce the need for animal and human tissue procurement, which is the current standard for ophthalmologic teaching.

Investigation of the antiviral activity of the recombinant human interferon lambda 1 in human conjunctiva cell culture

The aim of the study was to determine the efficacy of recombinant human interferon lambda 1 (IFN-λ1) against human adenovirus serotype 5 in a culture of human conjunctival cells Chang conjunctiva clone 1-5c-4. Material and methods. The study design consisted of three experimental schemes, reflecting a prophylactic and two options for a therapeutic and prophylactic treatment regimen (with the constant presence of the virus in the culture medium and with its removal after adsorption). The antiviral activity of IFN-λ1 was determined by the number of viable cells after exposure to the virus (MTT test). Results and discussion. It has been established that IFN-λ1 has antiviral activity against human adenovirus in vitro under a prophylactic and therapeutic-prophylactic scheme of administration at an infection dose of 1 and 10 TCID50 (50% tissue culture infectious dose), but not at an infection dose of 100 TCID50. The antiviral effect of the use of IFN-λ1 in a therapeutic and prophylactic regimen at an infection dose of 1 TCID50 was comparable to that of IFN-α. At the same time, both interferons did not have a toxic effect on the cell culture even at a concentration of 84 and 58 μg/ml, respectively. The antiviral activity and the absence of cytotoxic action provide the basis for further study of the possibility of development of based on IFN-λ1 drug for eye conjunctiva viral diseases treatment.