2016

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Gomes PJ, Lane KJ, Angjeli E, Stein L, Abelson MB. Technical and clinical validation of an environmental exposure unit for ragweed. J Asthma Allergy 2016;9:215-221.Abstract

BACKGROUND: Allergic rhinitis is a common condition, with ragweed pollen one of the more prevalent aeroallergens. Environmental exposure units such as the Allergen BioCube(®) are valuable models for clinical allergy studies. A study was conducted to validate the Allergen BioCube for uniform ragweed pollen concentrations and clinically relevant sign and symptom responses to ragweed exposure. METHODS: Ragweed pollen concentrations were measured on 3 consecutive days in the Allergen BioCube and verified by Rotorod collection and continuous laser particle count measurements. Subjects (N=10) were exposed to ragweed pollen in the BioCube for 3 hours per day for 3 consecutive days. Subjects assessed their nasal itching, sneezing, rhinorrhea, and nasal congestion during each BioCube exposure; total nasal symptom score was computed. Peak nasal inspiratory flow was also assessed during BioCube exposure. RESULTS: Uniform ragweed pollen concentrations were obtained throughout each of the 3-hour testing periods in the Allergen BioCube, both spatially and temporally, at all subject positions, with a low mean standard deviation of 10%. Pronounced increases in mean total nasal symptom scores (6.7±0.94 to 7.6±0.86, last 90 minutes of exposure) occurred for all three BioCube ragweed pollen exposure visits. Mean peak nasal inspiratory flow decreased 24% at 3 hours of BioCube exposure on Day 3. No safety issues of concern occurred in this study. CONCLUSION: The Allergen BioCube achieved technical and clinical validation for ragweed allergen. Ragweed pollen concentration was uniform both temporally and spatially. Allergic rhinitis signs and symptoms were induced in subjects during exposure to ragweed in the BioCube at clinically meaningful levels for allergy studies.

Gómez-Laberge C, Smolyanskaya A, Nassi JJ, Kreiman G, Born RT. Bottom-Up and Top-Down Input Augment the Variability of Cortical Neurons. Neuron 2016;91(3):540-7.Abstract

Neurons in the cerebral cortex respond inconsistently to a repeated sensory stimulus, yet they underlie our stable sensory experiences. Although the nature of this variability is unknown, its ubiquity has encouraged the general view that each cell produces random spike patterns that noisily represent its response rate. In contrast, here we show that reversibly inactivating distant sources of either bottom-up or top-down input to cortical visual areas in the alert primate reduces both the spike train irregularity and the trial-to-trial variability of single neurons. A simple model in which a fraction of the pre-synaptic input is silenced can reproduce this reduction in variability, provided that there exist temporal correlations primarily within, but not between, excitatory and inhibitory input pools. A large component of the variability of cortical neurons may therefore arise from synchronous input produced by signals arriving from multiple sources.

Gong Y, Fu Z, Edin ML, Liu C-H, Wang Z, Shao Z, Fredrick TW, Saba NJ, Morss PC, Burnim SB, Meng SS, Lih FB, Stephen Lee KS, Moran EP, SanGiovanni JP, Hellström A, Hammock BD, Zeldin DC, Smith LEH. Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization. Arterioscler Thromb Vasc Biol 2016;36(9):1919-27.Abstract

OBJECTIVE: Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. APPROACH AND RESULTS: The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. CONCLUSIONS: Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.

Gonzalez-Andrades M, Alonso-Pastor L, Mauris J, Cruzat A, Dohlman CH, Argüeso P. Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function. Sci Rep 2016;6:19395.Abstract

The repair of wounds through collective movement of epithelial cells is a fundamental process in multicellular organisms. In stratified epithelia such as the cornea and skin, healing occurs in three steps that include a latent, migratory, and reconstruction phases. Several simple and inexpensive assays have been developed to study the biology of cell migration in vitro. However, these assays are mostly based on monolayer systems that fail to reproduce the differentiation processes associated to multilayered systems. Here, we describe a straightforward in vitro wound assay to evaluate the healing and restoration of barrier function in stratified human corneal epithelial cells. In this assay, circular punch injuries lead to the collective migration of the epithelium as coherent sheets. The closure of the wound was associated with the restoration of the transcellular barrier and the re-establishment of apical intercellular junctions. Altogether, this new model of wound healing provides an important research tool to study the mechanisms leading to barrier function in stratified epithelia and may facilitate the development of future therapeutic applications.

Gonzalez-Gonzalez LA, Payal AR, Gonzalez-Monroy JE, Daly MK. Ophthalmic Surgical Simulation in Training Dexterity in Dominant and Nondominant Hands: Results From a Pilot Study. J Surg Educ 2016;73(4):699-708.Abstract

PURPOSE: To determine whether a structured training program using the validated EYESI surgical simulator improves dexterity in nondominant (ND) hands. SETTING: Academic tertiary referral center. DESIGN: Nonrandomized, prospective study. METHODS: Subjects who chose to participate and provided informed consent completed a structured simulation training program, which included a baseline test, 3 sessions of repeated tasks, and a final test on capsulorhexis in dominant (D) and ND hands. Participants completed demographic and satisfaction questionnaires. Performances at each session were recorded. We compared overall scores at baseline and at the end of the study, and analyzed trends over time. Statistical analysis was performed using JMP by SAS. RESULTS: Overall, 14 subjects completed the training program. In all, 3 (21.4%) were attending physicians and 11 (78.6%) were trainees. There was a significant improvement in the average overall scores (baseline vs. final) in both the D hand (33.4 vs. 46.5; p < 0.05) and the ND hand (28.9 vs. 47.7; p < 0.001). The structured training program demonstrated significantly faster performance times in both hands at the end of the study (D p< 0.001, ND p < 0.02). However, the learning curve was significantly steeper in the ND hand (p < 0.01). Participants agreed that simulation training improved the ND hand dexterity. CONCLUSIONS: We found a significantly greater trend for improvement in the ND compared with the D hand. These results suggest that an elaborate, structured curriculum targeting teaching dexterity results in better simulated performance.

Greenwald SH, Charette JR, Staniszewska M, Shi LY, Brown SDM, Stone L, Liu Q, Hicks WL, Collin GB, Bowl MR, Krebs MP, Nishina PM, Pierce EA. Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease. Am J Pathol 2016;186(7):1925-38.Abstract

The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.

Greiner JV. Long-Term (3 Year) Effects of a Single Thermal Pulsation System Treatment on Meibomian Gland Function and Dry Eye Symptoms. Eye Contact Lens 2016;42(2):99-107.Abstract

PURPOSE: The present study examined the long-term (3 years) effects of a single (12 min) thermal pulsation system (TPS) treatment on symptomatic patients with evaporative dry eye disease (DED) secondary to meibomian gland dysfunction (MGD). METHODS: In this prospective, cohort, observational, single-center study design, signs (meibomian gland secretion [MGS] scores and tear film breakup time [TBUT]) and symptoms (Ocular Surface Disease Index [OSDI] and Standard Patient Evaluation of Eye Dryness [SPEED] questionnaires) were determined in 20 patients (40 eyes) with MGD and dry eye symptoms at baseline (BL), 1 month, and 3 years post-TPS treatment using LipiFlow. RESULTS: Meibomian gland secretion scores increased from BL (4.5±0.8) to 1 month (12.0±1.1, P≤0.001). Improvement persisted at 3 years (18.4±1.4) relative to BL (P≤0.001). Meibomian gland secretion scores in all regions of the lower eyelid were improved over BL at 1 month (nasal [P≤0.001], central [P≤0.001], temporal [P≤0.01]) and 3 years (nasal [P≤0.001], central [P≤0.001], temporal [P≤0.001]). TBUT increased from BL (4.1±0.4) to 1 month (7.9±1.4, P≤0.05) but was not significantly different than BL at 3 years (4.5±0.6, P>0.05). The OSDI scores decreased from BL (26.0±4.6) to 1 month (14.7±4.3, P≤0.001) but returned to BL levels at 3 years (22.5±5.4, P>0.05). The SPEED scores decreased from BL (13.4±1.0) to 1 month (6.5±1.3, P≤0.001), and this improvement persisted at 3 years (9.5±1.6, P≤0.001). CONCLUSIONS: Thermal pulsation may be a uniquely efficacious treatment option for DED secondary to MGD in that a single 12-min procedure is associated with significant improvement in MGS and SPEED scores for up to 3 years.

Grishchuk Y, Stember KG, Matsunaga A, Olivares AM, Cruz NM, King VE, Humphrey DM, Wang SL, Muzikansky A, Betensky RA, Thoreson WB, Haider N, Slaugenhaupt SA. Retinal Dystrophy and Optic Nerve Pathology in the Mouse Model of Mucolipidosis IV. Am J Pathol 2016;186(1):199-209.Abstract

Mucolipidosis IV is a debilitating developmental lysosomal storage disorder characterized by severe neuromotor retardation and progressive loss of vision, leading to blindness by the second decade of life. Mucolipidosis IV is caused by loss-of-function mutations in the MCOLN1 gene, which encodes the transient receptor potential channel protein mucolipin-1. Ophthalmic pathology in patients includes corneal haze and progressive retinal and optic nerve atrophy. Herein, we report ocular pathology in Mcoln1(-/-) mouse, a good phenotypic model of the disease. Early, but non-progressive, thinning of the photoreceptor layer, reduced levels of rhodopsin, disrupted rod outer segments, and widespread accumulation of the typical storage inclusion bodies were the major histological findings in the Mcoln1(-/-) retina. Electroretinograms showed significantly decreased functional response (scotopic a- and b-wave amplitudes) in the Mcoln1(-/-) mice. At the ultrastructural level, we observed formation of axonal spheroids and decreased density of axons in the optic nerve of the aged (6-month-old) Mcoln1(-/-) mice, which indicates progressive axonal degeneration. Our data suggest that mucolipin-1 plays a role in postnatal development of photoreceptors and provides a set of outcome measures that can be used for ocular therapy development for mucolipidosis IV.

Grob SR, Yoon MK. Central Retinal Vein Occlusion Resolving After Orbital Decompression in Thyroid Eye Disease. Ophthal Plast Reconstr Surg 2016;Abstract

A 49-year-old male presented with proptosis and was found to have optic nerve edema with peripapillary hemorrhages. Diagnostic testing showed a suppressed thyroid-stimulating hormone. CT orbits showed homogenous tendon-sparing enlargement of the medial and inferior rectus muscles, characteristic of thyroid eye disease. Intravenous methylprednisolone was administered given the concern for compressive optic neuropathy. He initially had improvement of his symptoms, so orbital decompression was deferred. Subsequently he presented with worsening diplopia and right proptosis, a new afferent pupillary defect, and a cecocentral visual field defect. Dilated examination revealed significant optic nerve head edema and diffuse retinal hemorrhages in all 4 quadrants consistent with a central retinal vein occlusion. The patient underwent an urgent 3-wall orbital decompression on the right. Close follow up postoperatively showed resolution of the central retinal vein occlusion and the associated optic disc edema, peripapillary hemorrhages, and macular edema. Orbital decompression is known to improve many manifestations of thyroid eye disease, but this is the first report of orbital decompression resulting in resolution of a central retinal vein occlusion.

Gromova A, Voronov DA, Yoshida M, Thotakura S, Meech R, Dartt DA, Makarenkova HP. Lacrimal Gland Repair Using Progenitor Cells. Stem Cells Transl Med 2016;Abstract

: In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild-type mouse LGs by transplanting them into the LGs of TSP-1(-/-) mice, which represent a novel mouse model for ADDE. TSP-1(-/-) mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c-kit-positive epithelial cell adhesion molecule (EpCAM(+)) populations sorted from mouse LGs, the c-kit(+)dim/EpCAM(+)/Sca1(-)/CD34(-)/CD45(-) cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three-dimensional cultures. Moreover, when transplanted into injured or "diseased" LGs, they engraft into acinar and ductal compartments. EPCP-injected TSP-1(-/-) LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. SIGNIFICANCE: In humans, the lacrimal gland is the primary contributor to the aqueous layer of the tear film. Damage or inflammation of the lacrimal gland may lead to severe aqueous-deficiency dry eye and corneal disease. Endogenous lacrimal gland epithelial cell progenitors (EPCPs) injected into the gland of mouse model of human Sjögren's syndrome TSP-1(-/-) mice resulted in long-term engraftment and markedly improved structure and function of "diseased" lacrimal gland. This study demonstrates, for the first time, that EPCPs can mediate functional recovery of the lacrimal gland in a Sjögren's syndrome mouse model. These data establish proof of concept that endogenous stem/progenitor cell transplantation may be used to treat human lacrimal gland chronic inflammation.

Grotting LA, Davoudi S, Palenzuela D, Papaliodis GN, Sobrin L. Association of Low Vitamin D Levels With Noninfectious Anterior Uveitis. JAMA Ophthalmol 2016;Abstract

Importance: Vitamin D plays an important role in both the innate and adaptive immune systems. It has been shown to contribute to the etiology of T-cell-mediated autoimmune diseases through the upregulation of type 2 anti-inflammatory T helper cells and the suppression of type 1 T helper cells. Noninfectious uveitis is postulated to be caused by immune dysfunction. Objective: To determine whether there is an association between vitamin D levels and noninfectious anterior uveitis. Design, Setting, and Participants: This was a case-control study. We identified patients with and without noninfectious uveitis using the Massachusetts Eye and Ear Infirmary Ocular Inflammation Database and electronic medical records from March 1, 2008, to December 12, 2015, at the Massachusetts Eye and Ear Infirmary Uveitis and Comprehensive Ophthalmology Clinics. One hundred patients with noninfectious anterior uveitis and 100 patients without uveitis were recruited. Patients with noninfectious uveitis were diagnosed by fellowship-trained uveitis specialists after exclusion of infectious causes and neoplastic masquerades of uveitis. All patients included had a total 25-hydroxyvitamin D level recorded. Multivariate regression models were constructed to determine the association between vitamin D levels and the presence of uveitis. Main Outcome and Measure: Presence of noninfectious anterior uveitis. Results: We identified 100 patients (64 white, 8 African American, 25 Asian, and 3 Hispanic) with a mean (SD) age of 51.8 (15.9) years (26 men) and 100 control individuals (58 white, 23 African American, 8 Asian, and 11 Hispanic) with a mean (SD) age of 53.6 (16.2) years (27 men). Hypovitaminosis D was associated with noninfectious uveitis in the univariate analysis (odds ratio, 2.53; 95% CI, 1.42-4.51; P = .002). The association in multivariate regression after adjusting for age, sex, and race/ethnicity was 2.96 (95% CI, 1.60-5.50; P = .001) The odds of developing uveitis were 4% lower for every 1-ng/mL increase in vitamin D level (odds ratio, 0.96; 95% CI, 0.93-0.99; P = .01) in the main multivariate analysis. Conclusions and Relevance: In this retrospective study, lower vitamin D levels were associated with an increased risk of noninfectious anterior uveitis. However, this does not confirm a causal effect.

Gupta M, Jardeleza MSR, Kim I, Durand ML, Kim L, Lobo A-M. Varicella Zoster Virus Necrotizing Retinitis in Two Patients with Idiopathic CD4 Lymphocytopenia. Ocul Immunol Inflamm 2016;24(5):544-8.Abstract

PURPOSE: Progressive outer retinal necrosis (PORN) associated with varicella zoster virus (VZV) is usually diagnosed in HIV positive or immunosuppressed patients. We report two cases of immunocompetent patients with necrotizing viral retinitis found to have idiopathic CD4 lymphocytopenia. METHODS: Clinical presentation, examination, imaging, and laboratory testing of two patients with VZV retinitis are presented. RESULTS: An HIV negative patient with history of herpes zoster presented with rapid loss of vision and examination consistent with PORN. PCR testing confirmed VZV. Lymphocytopenia was noted with a CD4 count of 25/mm(3). A second HIV negative patient presented with blurred vision and lid swelling and was found to have peripheral VZV retinitis confirmed by PCR. Laboratory workup revealed lymphocytopenia with a CD4 count of 133/mm(3). CONCLUSIONS: VZV necrotizing retinitis classic for PORN can occur in HIV negative patients. Idiopathic CD4 lymphocytopenia should be considered healthy patients who develop ocular infections seen in the immunocompromised.

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Haleem MS, Han L, van Hemert J, Fleming A, Pasquale LR, Silva PS, Song BJ, Aiello LP. Regional Image Features Model for Automatic Classification between Normal and Glaucoma in Fundus and Scanning Laser Ophthalmoscopy (SLO) Images. J Med Syst 2016;40(6):132.Abstract

Glaucoma is one of the leading causes of blindness worldwide. There is no cure for glaucoma but detection at its earliest stage and subsequent treatment can aid patients to prevent blindness. Currently, optic disc and retinal imaging facilitates glaucoma detection but this method requires manual post-imaging modifications that are time-consuming and subjective to image assessment by human observers. Therefore, it is necessary to automate this process. In this work, we have first proposed a novel computer aided approach for automatic glaucoma detection based on Regional Image Features Model (RIFM) which can automatically perform classification between normal and glaucoma images on the basis of regional information. Different from all the existing methods, our approach can extract both geometric (e.g. morphometric properties) and non-geometric based properties (e.g. pixel appearance/intensity values, texture) from images and significantly increase the classification performance. Our proposed approach consists of three new major contributions including automatic localisation of optic disc, automatic segmentation of disc, and classification between normal and glaucoma based on geometric and non-geometric properties of different regions of an image. We have compared our method with existing approaches and tested it on both fundus and Scanning laser ophthalmoscopy (SLO) images. The experimental results show that our proposed approach outperforms the state-of-the-art approaches using either geometric or non-geometric properties. The overall glaucoma classification accuracy for fundus images is 94.4 % and accuracy of detection of suspicion of glaucoma in SLO images is 93.9 %.

Halilovic A, Schmedt T, Benischke A-S, Hamill C, Chen Y, Santos JH, Jurkunas UV. Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy. Antioxid Redox Signal 2016;24(18):1072-83.Abstract

AIMS: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. RESULTS: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. INNOVATION: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. CONCLUSION: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

Hattori T, Takahashi H, Dana R. Novel Insights Into the Immunoregulatory Function and Localization of Dendritic Cells. Cornea 2016;35 Suppl 1:S49-S54.Abstract

Dendritic cells (DCs) are antigen-presenting cells that normally play a critical role in stimulating T-cell-dependent immune responses. However, tolerogenic DCs (CD11cMHC-IICD80CD86) induce immune tolerance by stimulating regulatory T cells (Tregs: CD4CD25Foxp3). Although tolerogenic DCs are used to treat autoimmune diseases and to prevent transplantation rejection, the mechanisms by which they regulate alloimmunity are poorly understood. Here, we review our previous studies aiming to elucidate the mechanisms involved in immune rejection of corneal allografts using a corneal transplant model. We found that donor-derived tolerogenic DCs significantly prolonged corneal allograft survival by suppressing indirect allosensitization. We also reported the precise distribution of intraepithelial corneal DCs, termed Langerhans cells (LCs: CD11cLangerinMHC-II) in the cornea, which we maintain play a critical role in regulating corneal immunity. By confocal microscopy, we constructed 3-dimensional images of corneal LCs, which demonstrated that their cell bodies are present in the basal cell layer of the corneal epithelium. Furthermore, LC dendrites extend toward the ocular surface, but do not connect to epithelial tight junctions, indicating that they cannot directly interact with ocular surface antigens. We confirm the potential of DC therapy for corneal graft rejection and report the function of intraepithelial DCs (LCs) in the normal cornea.

He M, Storr-Paulsen T, Wang AL, Ghezzi CE, Wang S, Fullana M, Karamichos D, Utheim TP, Islam R, Griffith M, Islam MM, Hodges RR, Wnek GE, Kaplan DL, Dartt DA. Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion. Invest Ophthalmol Vis Sci 2016;57(14):6134-6146.Abstract

Purpose: We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods: We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results: Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions: Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study.

Hellgren G, Löfqvist C, Hård A-L, Hansen-Pupp I, Gram M, Ley D, Smith LE, Hellström A. Serum concentrations of vascular endothelial growth factor in relation to retinopathy of prematurity. Pediatr Res 2015;Abstract

BACKGROUND: The role of VEGF in the pathogenesis of retinopathy of prematurity (ROP) has been clearly established. However, little is known about temporal changes in circulating VEGF concentrations in the preterm infant. The objective was to determine the longitudinal serum concentrations of VEGF in relation to ROP. METHODS: This study included 52 infants born at <31 weeks gestational age (non-ROP n=33, non-proliferative ROP n=10, treated for ROP n=9). VEGF concentrations were analyzed in blood samples collected at birth, at 3 days postnatal age, and then weekly until at least a gestational age of 35 weeks. RESULTS: VEGF concentrations at birth did not differ between groups, independent of later ROP status. In contrast, VEGF serum concentrations were significantly higher at first detection of ROP in infants who were later treated for ROP compared to infants without ROP. At the time of laser therapy, serum VEGF concentrations did not differ between groups. CONCLUSION: Circulatory concentrations of VEGF, in infants who later developed severe ROP, were elevated at the time when ROP first was detected but not at the time when current treatment most often occurred. This supports the need for further studies of circulating VEGF in relation to the timing of ROP treatment.Pediatric Research (2015); doi:10.1038/pr.2015.181.

Hellström A, Ley D, Hansen-Pupp I, Hallberg B, Löfqvist C, van Marter L, van Weissenbruch M, Ramenghi LA, Beardsall K, Dunger D, Hård A-L, Smith LEH. Insulin-like growth factor 1 has multisystem effects on foetal and preterm infant development. Acta Paediatr 2016;105(6):576-86.Abstract

UNLABELLED: Poor postnatal growth after preterm birth does not match the normal rapid growth in utero and is associated with preterm morbidities. Insulin-like growth factor 1 (IGF-1) axis is the major hormonal mediator of growth in utero, and levels of IGF-1 are often very low after preterm birth. We reviewed the role of IGF-1 in foetal development and the corresponding preterm perinatal period to highlight the potential clinical importance of IGF-1 deficiency in preterm morbidities. CONCLUSION: There is a rationale for clinical trials to evaluate the potential benefits of IGF-1 replacement in very preterm infants.

Hellström A, Ley D, Hansen-Pupp I, Hallberg B, Ramenghi LA, Löfqvist C, Smith LEH, Hård A-L. Role of Insulinlike Growth Factor 1 in Fetal Development and in the Early Postnatal Life of Premature Infants. Am J Perinatol 2016;33(11):1067-71.Abstract

The neonatal period of very preterm infants is often characterized by a difficult adjustment to extrauterine life, with an inadequate nutrient supply and insufficient levels of growth factors, resulting in poor growth and a high morbidity rate. Long-term multisystem complications include cognitive, behavioral, and motor dysfunction as a result of brain damage as well as visual and hearing deficits and metabolic disorders that persist into adulthood. Insulinlike growth factor 1 (IGF-1) is a major regulator of fetal growth and development of most organs especially the central nervous system including the retina. Glucose metabolism in the developing brain is controlled by IGF-1 which also stimulates differentiation and prevents apoptosis. Serum concentrations of IGF-1 decrease to very low levels after very preterm birth and remain low for most of the perinatal development. Strong correlations have been found between low neonatal serum concentrations of IGF-1 and poor brain and retinal growth as well as poor general growth with multiorgan morbidities, such as intraventricular hemorrhage, retinopathy of prematurity, bronchopulmonary dysplasia, and necrotizing enterocolitis. Experimental and clinical studies indicate that early supplementation with IGF-1 can improve growth in catabolic states and reduce brain injury after hypoxic/ischemic events. A multicenter phase II study is currently underway to determine whether intravenous replacement of human recombinant IGF-1 up to normal intrauterine serum concentrations can improve growth and development and reduce prematurity-associated morbidities.

Hodges RR, Dartt DA. Signaling Pathways of Purinergic Receptors and Their Interactions with Cholinergic and Adrenergic Pathways in the Lacrimal Gland. J Ocul Pharmacol Ther 2016;32(8):490-497.Abstract

PURPOSE: Purinergic receptors play a key role in the function of the lacrimal gland (LG) as P1 purinergic receptors A1, A2A, and A2B, P2X1-7 receptors, and many of the P2Y receptors are expressed. METHODS: This review examines the current knowledge of purinergic receptors in the LG as well as the signaling pathways activated by these receptors. RESULTS: These receptors are expressed on the acinar, ductal, and myoepithelial cells. Considerable crosstalk exists between the pathways activated by P2X7 receptors with those activated by M3 muscarinic or α1D adrenergic receptors. The mechanism of the crosstalk between P2X7 and M3 muscarinic receptors differs from that of the crosstalk between P2X7 and α1D adrenergic receptors. CONCLUSIONS: Understanding purinergic receptors and how they modulate protein secretion could play a key role in normal and pathological responses of the LG.

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