Amparo F, Wang H, Emami-Naeini P, Karimian P, Dana R. The Ocular Redness Index: a novel automated method for measuring ocular injection. Invest Ophthalmol Vis Sci 2013;54(7):4821-6.Abstract
PURPOSE: To develop and validate a novel automated system to assess ocular redness (OR) in clinical images. METHODS: We developed a novel software that quantifies OR in digital images based on a mathematic algorithm using a centesimal continuous scoring scale. Subsequently, we conducted a study to validate the scores obtained with this system by correlating them with those obtained by two physicians using two image-based comparative subjective scales, the Efron and the Validated Bulbar Redness (VBR) grading scales. Additionally, we evaluated the level of clinical agreement between the Ocular Redness Index (ORI) score and the two image-based methods by means of the Bland-Altman analysis. Main outcome measures included correlation and level of agreement between the ORI score, Efron score, and the VBR score. RESULTS: One hundred and two clinical photographs of eyes with OR were evaluated. The ORI scores significantly correlated with the scores obtained by the two clinicians using the Efron (Observer 1, R=0.925, P<0.001; Observer 2, R=0.857, P<0.001), and VBR (Observer 1, R=0.830, P<0.001; Observer 2, R=0.821, P<0.001) scales. The Bland-Altman analysis revealed levels of disagreement of up to 30 and 27 units for the ORI-Efron and ORI-VBR score comparisons, respectively. CONCLUSIONS: The ORI provides an objective and continuous scale for evaluating ocular injection in an automated manner, and without need for a trained physician for scoring. The ORI may be used as a new alternative for objective OR evaluation in clinics and in clinical trials.
Ding J, Kam WR, Dieckow J, Sullivan DA. The influence of 13-cis retinoic acid on human meibomian gland epithelial cells. Invest Ophthalmol Vis Sci 2013;54(6):4341-50.Abstract
PURPOSE: Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells. METHODS: To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated. RESULTS: We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9. CONCLUSIONS: Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA-related induction of MGD.
Contreras-Ruiz L, Regenfuss B, Mir FA, Kearns J, Masli S. Conjunctival inflammation in thrombospondin-1 deficient mouse model of Sjögren's syndrome. PLoS One 2013;8(9):e75937.Abstract
Lacrimal gland inflammation during autoimmune Sjögren's syndrome (SS) leads to ocular surface inflammation - Keratoconjunctivitis sicca (KCS). This condition afflicts both the cornea and conjunctiva that form the ocular surface. Thrombospondin-1 (TSP-1) deficiency in mice results in lacrimal gland and corneal inflammation that resembles the human disease. In this study we report conjunctival pathology in this mouse model of SS. We found that TSP-1 null mice develop inflammation in the conjunctiva and associated loss of goblet cell function similar to that seen in patients with SS. Increased expression of Th1 (IFN-γ, TNF-α) and Th17 (IL-6, IL-17A) inflammatory cytokines and related transcription factors (Tbet and RORγt) were detected in TSP-1 null conjunctiva as well as their draining lymph nodes (LNs). The conjunctival inflammation was also accompanied by an increase in local lymphatic vessels. Interestingly, migration of antigen-bearing dendritic cells (DCs) from the ocular surface to the LNs was dependent on the TSP-1 available in the tissue. These results not only reveal potential immunopathogenic mechanisms underlying KCS in SS but also highlight the therapeutic potential of TSP-1.
Argüeso P. Glycobiology of the ocular surface: mucins and lectins. Jpn J Ophthalmol 2013;57(2):150-5.Abstract
Glycosylation is an important and common form of posttranscriptional modification of proteins in cells. During the last decade, a vast array of biological functions has been ascribed to glycans because of a rapid evolution in glycomic technologies. Glycogenes that are highly expressed at the human ocular surface include families of glycosyltransferases, proteoglycans, and glycan degradation proteins, as well as mucins and carbohydrate-binding proteins, such as the galectins. On the apical glycocalyx, mucin O-glycans promote boundary lubrication, prevent bacterial adhesion and endocytic activity, and maintain epithelial barrier function through interactions with galectins. The emerging roles attributed to glycans are contributing to the appreciation of their biological capabilities at the ocular surface.
Cruzat A, Tauber A, Shukla A, Paschalis EI, Pineda R, Dohlman CH. Low-cost and readily available tissue carriers for the Boston keratoprosthesis: a review of possibilities. J Ophthalmol 2013;2013:686587.Abstract
The Boston keratoprosthesis (B-KPro), currently the most commonly used artificial cornea worldwide, can provide rapid visual rehabilitation for eyes with severe corneal opacities not suitable for standard corneal transplantation. However, the B-KPro presently needs a corneal graft as a tissue carrier. Although corneal allograft tissue is readily available in the United States and other developed countries with established eye banks, the worldwide need vastly exceeds supply. Therefore, a simple, safe, and inexpensive alternative to corneal allografts is desirable for the developing world. We are currently exploring reasonable alternative options such as corneal autografts, xenografts, noncorneal autologous tissues, and laboratory-made tissue constructs, as well as modifications to corneal allografts, such as deep-freezing, glycerol-dehydration, gamma irradiation, and cross-linking. These alternative tissue carriers for the B-KPro are discussed with special regard to safety, practicality, and cost for the developing world.
Chen Y, Chauhan SK, Lee HS, Stevenson W, Schaumburg CS, Sadrai Z, Saban DR, Kodati S, Stern ME, Dana R. Effect of desiccating environmental stress versus systemic muscarinic AChR blockade on dry eye immunopathogenesis. Invest Ophthalmol Vis Sci 2013;54(4):2457-64.Abstract
PURPOSE: A majority of experimental data on dry eye disease (DED) immunopathogenesis have been derived from a murine model of DED that combines desiccating environmental stress with systemic muscarinic acetylcholine receptor (mAChR) inhibition. However, to our knowledge the effects of pharmacologic mAChR blockade on the pathogenesis of experimental DED have not been evaluated systemically. The purpose of our study was to investigate the differential effects of desiccating environmental stress and mAChR inhibition on the pathogenesis of DED. METHODS: DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled-environment chamber or to systemic scopolamine, or by performing extraorbital lacrimal gland excision. Clinical disease was assessed using corneal fluorescein staining (CFS) and the cotton thread test (CTT). Corneal CD11b(+) and conjunctival CD3(+) T-cell infiltration were evaluated by flow cytometry. T-cells from draining cervical lymph nodes (CLN) and distant inguinal lymph nodes (ILN) were analyzed for Th1, Th2, Th17, and Treg responses by flow cytometry and ELISA. RESULTS: Desiccating environmental stress and systemic mAChR blockade induced similar clinical signs of DED. However, desiccating environmental stress imparted higher conjunctival CD3(+) T-cell infiltration, and greater Th17-cell activity and Treg dysfunction than mAChR blockade, while mAChR blockade decreased tear secretion to a greater extent than desiccating environmental stress. Systemic mAChR blockade attenuated Th17 activity and enhanced Th2 and Treg responses without affecting Th1 activity. CONCLUSIONS: In vivo inhibition of mAChRs variably affects CD4(+) T-cell subsets, and desiccating environmental stress and systemic mAChR blockade induce DED through different primary pathogenic mechanisms.
Amparo F, Dastjerdi MH, Okanobo A, Ferrari G, Smaga L, Hamrah P, Jurkunas U, Schaumberg DA, Dana R. Topical interleukin 1 receptor antagonist for treatment of dry eye disease: a randomized clinical trial. JAMA Ophthalmol 2013;131(6):715-723.Abstract
IMPORTANCE: The immunopathogenic mechanisms of dry eye disease (DED), one of the most common ophthalmic conditions, is incompletely understood. Data from this prospective, double-masked, randomized trial demonstrate that targeting interleukin 1 (IL-1) by topical application of an IL-1 antagonist is efficacious in significantly reducing DED-related patient symptoms and corneal epitheliopathy. OBJECTIVE: To evaluate the safety and efficacy of treatment with the topical IL-1 receptor antagonist anakinra (Kineret; Amgen Inc) in patients having DED associated with meibomian gland dysfunction. DESIGN AND SETTING: Prospective phase 1/2, randomized, double-masked, vehicle-controlled clinical trial. PARTICIPANTS: Seventy-five patients with refractory DED. INTERVENTIONS: Participants were randomized to receive treatment with topical anakinra, 2.5% (n = 30), anakinra, 5% (n = 15), or vehicle (1% carboxymethylcellulose) (n = 30) 3 times daily for 12 weeks. MAIN OUTCOMES AND MEASURES: Primary outcomes were corneal fluorescein staining (CFS), complete bilateral CFS clearance, dry eye-related symptoms as measured by the Ocular Surface Disease Index, tear film breakup time, and meibomian gland secretion quality. RESULTS: Topical anakinra was well tolerated compared with vehicle, with no reports of serious adverse reactions attributable to the therapy. After 12 weeks of therapy, participants treated with anakinra, 2.5%, achieved a 46% reduction in their mean CFS score (P = .12 compared with vehicle and P < .001 compared with baseline); participants treated with anakinra, 5%, achieved a 17% reduction in their mean CFS score (P = .88 compared with vehicle and P = .33 compared with baseline); and patients treated with vehicle achieved a 19% reduction in their mean CFS score (P = .11). Complete bilateral CFS clearance was noted in 8 of 28 patients (29%) treated with anakinra, 2.5%, vs in 2 of 29 patients (7%) treated with vehicle (P = .03). By week 12, treatment with anakinra, 2.5%, and treatment with anakinra, 5%, led to significant reductions in symptoms of 30% and 35%, respectively (P = .02 and P = .01, respectively, compared with vehicle); treatment with vehicle led to a 5% reduction in symptoms. CONCLUSIONS AND RELEVANCE: Treatment with topical anakinra, 2.5%, for 12 weeks was safe and significantly reduced symptoms and corneal epitheliopathy in patients with DED. These data suggest that the use of an IL-1 antagonist may have a role as a novel therapeutic option for patients with DED. TRIAL REGISTRATION Identifier: NCT00681109.
Contreras-Ruiz L, Ghosh-Mitra A, Shatos MA, Dartt DA, Masli S. Modulation of conjunctival goblet cell function by inflammatory cytokines. Mediators Inflamm 2013;2013:636812.Abstract
Ocular surface inflammation associated with Sjögren's syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren's syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-α and IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.
Amparo F, Sadrai Z, Jin Y, Alfonso-Bartolozzi B, Wang H, Shikari H, Ciolino JB, Chodosh J, Jurkunas U, Schaumberg DA, Dana R. Safety and efficacy of the multitargeted receptor kinase inhibitor pazopanib in the treatment of corneal neovascularization. Invest Ophthalmol Vis Sci 2013;54(1):537-44.Abstract
PURPOSE: To evaluate the safety and efficacy of topical pazopanib in the treatment of corneal neovascularization (CNV). METHODS: Twenty eyes of 20 patients with stable CNV were enrolled in a prospective, open label, noncomparative study and treated with topical pazopanib 0.5% for 3 weeks, and followed for 12 weeks. The primary endpoint was to determine the tolerability and safety of topical pazopanib in the treatment of CNV defined by the occurrence of ocular and systemic adverse events during the study. The secondary endpoint was to evaluate the effect of topical pazopanib on the reduction of (1) neovascular area (NA), defined as the area of the corneal vessels themselves, (2) invasion area (IA), defined as the fraction of the total cornea into which the vessels extend, (3) vessel length (VL), defined as the mean measurement of the extent of vessels from end to end, and (4) vessel caliber (VC), defined as the mean diameter of the corneal vessels. RESULTS: There were no severe adverse events following the use of topical pazopanib. Compared with the baseline visit, NA and VL showed a statistically significant decrease at week 3 (P = 0.02 and 0.01, respectively); and NA, IA, and VL statistically significantly decreased at week 12 (P = 0.03, 0.04, and <0.01, respectively). Visual acuity maintained without changes after the 12 week follow-up. CONCLUSIONS: This preliminary study suggests that topical treatment with pazopanib 0.5% is safe, well tolerated, and may have a role as an alternative for the treatment of CNV ( number, NCT01257750).
Yamaguchi T, Turhan A, Harris DL, Hu K, Prüss H, von Andrian U, Hamrah P. Bilateral nerve alterations in a unilateral experimental neurotrophic keratopathy model: a lateral conjunctival approach for trigeminal axotomy. PLoS One 2013;8(8):e70908.Abstract
To study bilateral nerve changes in a newly developed novel mouse model for neurotrophic keratopathy by approaching the trigeminal nerve from the lateral fornix. Surgical axotomy of the ciliary nerve of the trigeminal nerve was performed in adult BALB/c mice at the posterior sclera. Axotomized, contralateral, and sham-treated corneas were excised on post-operative days 1, 3, 5, 7 and 14 and immunofluorescence histochemistry was performed with anti-β-tubulin antibody to evaluate corneal nerve density. Blink reflex was evaluated using a nylon thread. The survival rate was 100% with minimal bleeding during axotomy and a surgical time of 8±0.5 minutes. The blink reflex was diminished at day 1 after axotomy, but remained intact in the contralateral eyes in all mice. The central and peripheral subbasal nerves were not detectable in the axotomized cornea at day 1 (p<0.001), compared to normal eyes (101.3±14.8 and 69.7±12.0 mm/mm² centrally and peripherally). Interestingly, the subbasal nerve density in the contralateral non-surgical eyes also decreased significantly to 62.4±2.8 mm/mm² in the center from day 1 (p<0.001), but did not change in the periphery (77.3±11.7 mm/mm², P = 0.819). Our novel trigeminal axotomy mouse model is highly effective, less invasive, rapid, and has a high survival rate, demonstrating immediate loss of subbasal nerves in axotomized eyes and decreased subbasal nerves in contralateral eyes after unilateral axotomy. This model will allow investigating the effects of corneal nerve damage and serves as a new model for neurotrophic keratopathy.
Ziaei A, Schmedt T, Chen Y, Jurkunas UV. Sulforaphane decreases endothelial cell apoptosis in fuchs endothelial corneal dystrophy: a novel treatment. Invest Ophthalmol Vis Sci 2013;54(10):6724-34.Abstract
PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is an oxidative stress disorder that leads to age-related and gradual loss of corneal endothelial cells resulting in corneal edema and loss of vision. To date, other than surgical intervention, there are no treatment options for patients with FECD. We have shown that in FECD, there is a deficiency in nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated antioxidant defense due to decreased Nrf2 nuclear translocation and activation of antioxidant response element (ARE). In this study, we used sulforaphane (SFN) and D3T to investigate a strategy of targeting Nrf2-ARE in FECD. METHODS: FECD and normal ex vivo corneas and human corneal endothelial cell lines were pretreated with SFN or D3T and exposed to oxidative stress with tert-Butyl hydroperoxide (tBHP). Apoptosis was detected with TUNEL. Cellular localization of Nrf2 and p53 was assessed by immunohistochemistry. Effect of SFN was determined by using DCFDA assay, Western blot and real-time PCR. RESULTS: After pretreatment with SFN, oxidative stress was induced with tBHP. In ex vivo FECD specimens, SFN decreased CEC apoptosis by 55% in unstressed group and by 43% in tBHP-treated specimens. SFN enhanced nuclear translocation of Nrf2 in FECD specimens and decreased p53 staining under oxidative stress. Pretreatment with SFN enhanced cell viability by decreasing intracellular reactive oxygen species production. Upregulation of Nrf2 levels led to increased synthesis of DJ-1, heme oxygenase 1, and nicotinamide adenine dinucleotide quinone oxidoreductase-1. SFN significantly upregulated major ARE-dependent antioxidants and ameliorated oxidative stress-induced apoptosis in FECD. CONCLUSIONS: Our results suggest that targeting Nrf2-ARE pathway may arrest degenerative cell loss seen in FECD.
Spurr-Michaud SJ, Gipson IK. Methods for culture of human corneal and conjunctival epithelia. Methods Mol Biol 2013;945:31-43.Abstract
The surface of the eye is exposed to the outside world and is, thus, subject to surface abrasion, infections, and drying, cicatrizing diseases. Availability of in vitro methods for culture of the human corneal and conjunctival epithelia, which cover the ocular surface, is therefore important in understanding the biology of these epithelia and their response to disease/infections, as well as for providing human-relevant models for preclinical testing of potential therapeutic agents. The ensuing chapter describes several methods for primary culture of both corneal and conjunctival epithelia and culture of immortalized cell lines, and methods employed to induce differentiation in the cultured epithelia.
Woodward AM, Mauris J, Argüeso P. Binding of transmembrane mucins to galectin-3 limits herpesvirus 1 infection of human corneal keratinocytes. J Virol 2013;87(10):5841-7.Abstract
Epithelial cells lining mucosal surfaces impose multiple barriers to viral infection. At the ocular surface, the carbohydrate-binding protein galectin-3 maintains barrier function by cross-linking transmembrane mucins on the apical glycocalyx. Despite these defense mechanisms, many viruses have evolved to exploit fundamental cellular processes on host cells. Here, we use affinity assays to show that herpes simplex virus type 1 (HSV-1), but not HSV-2, binds human galectin-3. Knockdown of galectin-3 in human corneal keratinocytes by small interfering RNA significantly impaired HSV-1 infection, but not expression of nectin-1, indicating that galectin-3 is a herpesvirus entry mediator. Interestingly, exposure of epithelial cell cultures to transmembrane mucin isolates decreased viral infectivity. Moreover, HSV-1 failed to elute the biological counterreceptor MUC16 from galectin-3 affinity columns, suggesting that association of transmembrane mucins to galectin-3 provides protection against viral infection. Together, these results indicate that HSV-1 exploits galectin-3 to enhance virus attachment to host cells and support a protective role for transmembrane mucins under physiological conditions by masking viral entry mediators on the epithelial glycocalyx.
Yousuf MA, Zhou X, Mukherjee S, Chintakuntlawar AV, Lee JY, Ramke M, Chodosh J, Rajaiya J. Caveolin-1 associated adenovirus entry into human corneal cells. PLoS One 2013;8(10):e77462.Abstract
The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.