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ARVO 2014 Annual Meeting Abstracts 283 Autoimmune Ocular Disease and Allergy Monday, May 05, 2014 3:45 PM–5:30 PM Exhibit/Poster Hall SA Poster Session Program #/Board # Range: 2481–2520/C0001–C0040 Organizing Section: Immunology/Microbiology Program Number: 2481 Poster Board Number: C0001 Presentation Time: 3:45 PM–5:30 PM A model of allergic conjunctivitis in new zealand rabbits using ragweed pollen Paloma A. Marquez, Oscar Olvera, Yussett Contreras, Paulina Melgarejo, Leopoldo M. Baiza-Duran, Jonathan Bonilla, Gabriela Fregoso. pre-clinical, Laboratorios Sophia, Zapopan, Mexico. Purpose: To develop a reproducible model of ocular allergy in new zealand rabbits by assessing allergy clinical signs correlating IgE by immunohistochemistry. Methods: preclinical, prospective, longitudinal study. The study subjects were 5 healthy male albino new zealand rabbits treated under laboratory conditions and according to ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, these research subjects were enumerated from 1 to 5 for its study. The allergen used was ragweed pollen extract standardized high power (ambrosia trifida). 100 mg were injected intraperitoneally three times during the study on days 1, 8 and 15, on day 21 allergen challenge was performed applying 400ug into the conjunctival sac. The clinical review was performed by biomicroscopy and the following signs of allergy were evaluated: conjunctival hyperemia, eyelid edema, conjunctival edema and increased tear film, was performed on days 0 (baseline ), 21 ( 20 minutes after allergen challenge ), 22, 25 28 and 31; serum IgE levels were determined by immunohistochemistry using ELISA Kit mybiosource, the blood sample were taken on days 0, 8, 15, 21 (20 minutes after allergen challenge ), 25, 28 and 31. Results: On day 21 conjunctival hyperemia, eyelid edema, conjunctival edema and increase in tear film were observed in a moderate grade, on days 22, 25 and 28 these signs were mild and on day 31 were absent. The mean concentration of serum IgE was 5.7pg/ ml baseline in subsequent measurements to the allergen challenge showed a mean concentration of 8.2pg/ml, 7.4pg/ml, 7.1pg/ml and 6.6 pg / ml on day 21, 25, 28 and 31 respectively; being statistically significant (p <0.05) compared with the baseline measurement on day 21. Conclusions: In our model we found a relationship between clinical signs of allergy and IgE mediated immune response, however did not submit the severity reported in other allergic animal models in efficacy studies. It will be necessary to modify the dose of intraperitoneal sensitization in subsequent studies. Commercial Relationships: Paloma A. Marquez, Laboratorios Sophia (E); Oscar Olvera, Laboratorios Sophia (E); Yussett Contreras, Laboratorios Sophia (E); Paulina Melgarejo, Laboratorios Sophia (E); Leopoldo M. Baiza-Duran, Laboratorios Sophia (E); Jonathan Bonilla, Laboratorios Sophia (E); Gabriela Fregoso, Laboratorios Sophia (E) Support: Laboratorios Sophia SA de CV Program Number: 2482 Poster Board Number: C0002 Presentation Time: 3:45 PM–5:30 PM The third generation of antihistamines: Assessment of Histamine H1/H4 Receptor Antagonists in a Murine Model of Allergic Conjunctivitis Matt J. Chapin1, Laura Belen1, Andy Whitlock1, Jac Wijkmans2, Tiffany van de Meer2, Mounir Andaloussi2, Rogier A. Smits2, Iwan de Esch2, 3, Rob Leurs2, 3. 1ORA, Andover, MA; 2Griffin Discoveries BV, Amsterdam, Netherlands; 3Medicinal Chemistry, Faculty of Exact Sciences, VU University Amsterdam, Amsterdam, Netherlands. Purpose: This study tested the hypothesis that drugs antagonizing both the H1 and the H4 histamine receptors (H1R and H4R) may provide superior relief for the signs and symptoms of allergic conjunctivitis when compared to traditional allergy therapies. Methods: The study employed a murine conjunctival allergen challenge (CAC) model to test two structurally unrelated dual-action H1R/H4R antagonists: GD134 and GD136, as well as selective H4R antagonist GD135. Two positive comparators (olopatadine and prednisolone) and a vehicle control were also tested. Test articles were masked to investigators for the duration of the study. Animals (female balb/c mice) were sensitized by injection with a short ragweed allergen (SRW)/adjuvant mixture, and then challenged with topical SRW 17 days later to confirm an allergic response. Nine animals were randomly assigned to each of the 6 treatment groups. Animals received topical applications of test agent on 3 consecutive days, and then underwent CAC twice daily for 4 successive days; test agents were also applied on these days. Mean group values for hyperemia and squinting were compared with vehicle and with baseline to determine test agent effects. Results: Positive comparators reduced post-CAC hyperemia scores relative to the vehicle controls; these reductions were significant for prednisolone at challenge 1, 4, 6 and 8 (p <0.05). Both H1R/ H4R antagonists reduced hyperemia significantly (GD136, 3 of 4 challenges significant at p<0.05; GD134, 4 challenges significantly lower at p<0.05). Selective H4R antagonist GD135 did not significantly reduce hyperemia. Squinting scores exhibited a progressive decline with each successive challenge, however only the scores for olopatadine at challenge 8 were significantly reduced when compared to controls (p<0.05). Conclusions: Current antihistamine therapy for allergic conjunctivitis focuses on antagonism of the H1R signaling pathway. Despite this, recent studies have revealed not only a role for H4 receptors in the etiology of itch, the hallmark symptom of ocular allergy, but also identified the H4R as a new target to treat inflammation. Our studies provide evidence that two of our test compounds, GD134 and GD136, may have potential efficacy in the treatment of allergic conjunctivitis. Future studies, such as dose-ranging trials or studies in humans will help to clarify their potential. Commercial Relationships: Matt J. Chapin, Ora, Inc (E); Laura Belen, Ora, Inc (E); Andy Whitlock, Ora, Inc (E); Jac Wijkmans, Griffin Discoveries BV (E), Griffin Discoveries BV (P); Tiffany van de Meer, Griffin Discoveries BV (E), Griffin Discoveries BV (P); Mounir Andaloussi, Griffin Discoveries BV (E), Griffin Discoveries BV (P); Rogier A. Smits, Griffin Discoveries BV (I), Griffin Discoveries BV (P); Iwan de Esch, Griffin Discoveries BV (I), Griffin Discoveries BV (P); Rob Leurs, Griffin Discoveries BV (I), Griffin Discoveries BV (P) ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Program Number: 2483 Poster Board Number: C0003 Presentation Time: 3:45 PM–5:30 PM Automated Assessment of Conjunctival White Cell Infiltration by Confocal Microscopy Endri Angjeli, Colleen Heckley, Paul J. Gomes, Keith J. Lane. R&D, Ora, Inc, Andover, MA. Purpose: To develop an automated approach for the quantification of inflammatory cell infiltration into the conjunctiva in the course of an allergic inflammatory event. Methods: The study examined allergen challenge responses in subjects with a history of sensitivity to dust mites using a doublemasked, randomized, placebo-controlled protocol. Tears natural II and Prednisolone Phosphate were used as placebo and active test compounds. Entry criteria included both minimal response to allergen challenge and maximal response to a saline (sham) challenge. Test CAC was conducted following 5 days QID dosing of either placebo or active. Evaluation criteria included (1) the number of white cells within the vessel, (2) number of white cells on the vessel walls, and (3) the number of cells in the tissue surrounding the vessel per unit area, following a challenge. There were 13 subjects who completed the study. An algorithm developed using Image J software was used to quantify cells in images captured using an HRT II corneal module confocal microscope. Images were captured immediately pre- and post-challenge, and again at 16 hours post-challenge. Results: Counts of cells for all 3 areas of interest were elevated in placebo subjects compared to prednisolone-treated eyes; this difference was statistically significant for cells apparently adhered to the vessel walls at the 16 hour time point. Post-CAC time course of these measures showed that this difference develops for many hours following allergen challenge, demonstrating a chronic response which was prevented by the topical steroid. Conclusions: This study demonstrated that delayed time-points such as the 16 hour post-CAC measure may be critical to the use of automated methods going forward. Future efforts may employ time course measures in which inflammation can be tracked from imaging of white cells adhering to vessel walls to the process of diapedesis, and subsequent dispersal of cells into surrounding tissues. Commercial Relationships: Endri Angjeli, Ora, Inc (E); Colleen Heckley, Ora, Inc (E); Paul J. Gomes, Ora, Inc (E); Keith J. Lane, Ora, Inc (E) Clinical Trial: NCT01730872 Program Number: 2484 Poster Board Number: C0004 Presentation Time: 3:45 PM–5:30 PM Enhanced expression of conjunctival IL-9 co-localized with mast cells in seasonal allergic conjunctivitis and its effect on mast cell cytokine secretion via IL-9R Amirah Mohd Zaki, Grazyna Galatowicz, Virginia L. Calder. Department of Ocular Biology & Therapeutics, UCL Institute of Opthalmology, London, United Kingdom. Purpose: Mast cells (MC) are well known as the primary responders in allergy, secreting pro-inflammatory mediators including cytokines into the extracellular environment. IL-9 is a pro-inflammatory cytokine that is associated with the immunopathogenesis of allergic diseases including asthma. However, its role in allergic conjunctivitis is still unknown. The aim of this study was to investigate conjunctival expression of IL-9, and its role in cytokine secretion by mast cells. Methods: Human anonymised conjunctival tissue biopsies (3μm) were obtained from SAC donors (n=8, from all males; age: 18-65 years) at 8 hours’ post allergen challenge, and normal, non-inflamed, conjunctival tissues from anonymised donors (n=8 from 3 males; age 31-57 years). Donor tissues were collected after obtaining informed consent and Local Ethics approval in accordance with the Declaration of Helsinki. Sequential tissue sections were stained for anti-human IL-9 (Abcam), anti-human MC tryptase (AA1;DAKO) and primary antibody was omitted as a negative control. Two independent, masked observers enumerated positively stained cells per biopsy area (at least three fields). To study the function of IL-9 in vitro, bone marrow derived murine mouse mast cells (BMMCs) were exposed to PMA/ionomycin, ionomycin alone or anti-IgE in the presence or without anti-IL-9 or anti-IL-9R antibodies (R&D). Cytokine secretion at 24, 48, 72, 96, and 120 hours was assayed using multiplex bead arrays (Luminex). Results: IL-9 expressing cells were detected mainly within the subepithelial and stromal areas. There was a significant increase in numbers of IL-9+ cells in SAC tissues (mean=10.25 ± 1.26) compared to controls (mean= 25.00 ± 3.72; P<0.01). MC numbers were increased in SAC tissues and co-localised with IL-9. IL-9 was detected 24 hours’ post stimulation and reached its maximum at 48 hr in response to PMA/ionomycin and anti-IgE and 96 hr in response to ionomycin. Following neutralization of IL-9, secretion of IL-4, IL-5 and IL-13 was upregulated whereas these cytokine levels decreased upon blocking IL-9R (P<0.05). Conclusions: IL-9 expression within conjunctival tissues was upregulated during challenge and is secreted by mast cells. In vitro studies revealed that IL-9 affects IL-4, IL-5 and IL-13 secretion from BMMC. Commercial Relationships: Amirah Mohd Zaki, None; Grazyna Galatowicz, None; Virginia L. Calder, None Program Number: 2485 Poster Board Number: C0005 Presentation Time: 3:45 PM–5:30 PM Dendritic Cell-produced IL-33 Links Microbial Pathogens to Allergic Inflammation De-Quan Li1, Zhitao Su1, 2, Jing Lin1, 3, Lili Zhang1, Cintia S. De Paiva1, Stephen C. Pflugfelder1. 1Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX; 2School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China; 3Ophthalmology, Affiliated Hospital of Qingdao University Medical College, Qingdao, China. Purpose: Interleukin-33 (IL-33) has been identified as a novel proallergic cytokine that initiate Th2-dominant allergic inflammation. Dendritic cells (DCs) are major innate immunity cells; however, it is not clear whether DCs produce IL-33. This study identified a novel mechanism by which DCs links microbial pathogens to allergic inflammation via Toll-like receptor (TLR), NF-κB and IL-33 signaling pathway. Methods: Mouse bone marrow derived DCs were treated with or without microbial pathogens or recombinant mouse (rm) IL-33. The mRNA expression was determined by reverse transcription and real time PCR, and protein production was evaluated by ELISA, Western blotting, immunofluorescent staining and flow cytometry. Results: IL-33 mRNA and protein were found to be expressed by DCs and largely induced by specific microbial pathogens, especially lipopolysaccharide (LPS) and flagellin, the ligands to TLR4 and TLR5 respectively. Using a mouse model of topical challenge by LPS and flagellin, IL-33-producing DCs were observed in ocular mucosal surface and the draining cervical lymph nodes. LPS and flagellin were found to promote DC maturation with enhanced expression of CD40, CD80, CD86 and MHC classs II. The increased expression of MyD88, NF-κB1, NF-κB2 and RelA accompanied by NF-κB p65 activation with nuclear translocation was observed in DCs exposed to flagellin. The IL-33 induction by flagellin was significantly blocked by TLR5 antibody or NFκB activation inhibitor quinazoline. When treated with rmIL-33, DCs were activated to express the increased maturation markers CD40 and CD80, stimulated Th2 (IL-4, IL-5 and ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts IL-13) and inflammatory mediators (TNFα, IL-1β and CCL17), as well as OX40L that enables amplification of Th2 cell differentiation. This stimulatory effect of IL-33 in DCs was significantly blocked by ST2 antibody or soluble ST2. Conclusions: These findings demonstrate that DCs produce IL-33 via TLR/NF-κB signaling pathways, suggesting a molecular mechanism by which local allergic inflammatory response may be amplified by DC-produced IL-33. Commercial Relationships: De-Quan Li, None; Zhitao Su, None; Jing Lin, None; Lili Zhang, None; Cintia S. De Paiva, None; Stephen C. Pflugfelder, None Support: National Institutes of Health grant EY11915 (SCP), an unrestricted grant from Research to Prevent Blindness, the Oshman Foundation and the William Stamps Farish Fund. Program Number: 2486 Poster Board Number: C0006 Presentation Time: 3:45 PM–5:30 PM Role of Chemokine C-C motif ligand 7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis Chuan-Hui Kuo1, Andrea Collins1, Santa J. Ono1, 2. 1Allergy & Immunology, Cincinnati Children’s Hospital, Cincinnati, OH; 2 University of Cincinnati, Cincinnati, OH. Purpose: Molecules that are necessary for ocular hypersensitivity reactions include the chemokine receptors CCR1 and CCR3; chemokine C-C motif ligand 7 (CCL7) is a genetic target and ligand for these receptors. Therefore, we hypothesized that CCL7 mediates ocular hypersensitivity reactions and investigated CCL7’s necessity in a murine model of IgE-mediated allergic conjunctivitis using wildtype (WT) and CCL7-deficient mice. Methods: Allergic conjunctivitis was induced in WT and CCL7deficient mice. Mice were sensitized with OVA/alum intraperitoneally and subjected to OVA-induced ocular anaphylaxis via eye drops. Early-phase reactions including clinical symptoms, gene expressions and pathological changes in the conjunctival tissue were evaluated. Bone marrow–derived mast cells (BMMCs) from WT and CCL7deficient mice were sensitized and activated. β-hexosaminidase activity was measured by incubating the supernatants with p-nitrophenyl N-acetyl-β-D- glucosaminide. Results: The early-phase clinical symptoms in the conjunctiva after OVA challenge were significantly higher in OVA-sensitized than in vehicle-sensitized WT mice, but this OVA-induced increase was suppressed in CCL7-deficient mice. The mRNAs for FcεRIα and the connective tissue-type mast cell proteases were detected in the conjunctiva of both OVA-induced WT and CCL7-deficient mice. Notably, in OVA-induced anaphylaxis, the number of conjunctival mast cells was lower in CCL7-deficient than in WT mice, and BMMCs from CCL7-deficient mice showed decreased degranulation with IgE and antigen treatment compared with BMMCs from WT mice. Conclusions: Our results demonstrate that chemokine CCL7 deficiency suppressed OVA-induced ocular anaphylaxis, including regulating mast cell recruitment in vivo and reducing FcεRI-mediated mast cell activation in vitro. A better understanding of CCL7’s roles in mediating ocular hypersensitivity reactions will provide insights into mast cell function and novel treatments for allergic ocular diseases. Commercial Relationships: Chuan-Hui Kuo, None; Andrea Collins, None; Santa J. Ono, None Support: NIH R01-EY-019630-01 Program Number: 2487 Poster Board Number: C0007 Presentation Time: 3:45 PM–5:30 PM Airborne Particulate Matter (PM2.5) and Prevalence of Allergic Conjunctivitis in Japan Tatsuya Mimura, Masao Matsubara. Department of Ophthalmology, Tokyo Women’s Med Univ Med Ctr East, Arakawa-ku, Japan. Purpose: Exposure to particulate matter less than 2.5 μm in aerodynamic diameter (PM2.5) was associated with increasing severity of asthma and respiratory symptoms; however, little is known about the effect of PM2.5 with allergic conjunctivitis. The purpose of this study is to examine the associations of PM2.5 on the daily clinical visits for allergic conjunctivitis. Methods: We conducted a time-series analysis of associations between outpatient visits for allergic conjunctivitis and PM2.5 concentrations during May to July (non-pollen season) and August to October 2012 (autumnal pollen season). Air pollution data including PM2.5, oxidant, nitric oxide, nitrogen dioxide, nitrogen oxide, carbon monoxide, methane, non-methane hydrocarbons, and total hydrocarbons, and daily weather conditions (temperatures, wind speed and humidity) were collected at a centrally located monitor in Tokyo. We calculated weekly averages for the daily number of outpatient visits and data of air pollution and weather conditions and used the weekly average data for the analysis. Results: The only significant association obtained was between outpatient visits for allergic conjunctivitis and the concentration of PM2.5 (r=0.62, p=0.0177) during May to July, while, no correlation was found between the number of outpatients and any variables of air conditions during August to November. Multivariate analysis also showed that significant predictor of the number of outpatients was the concentration of PM2.5 during May to July (odds ratio=9.05, p=0.0463), while there were no significant predictors of the number of outpatients during August to October. During May to July, PM2.5 was negatively correlated with humidity (r=-0.53, p=0.0499). Conclusions: These results suggest a possible role for PM2.5 in the development of allergic conjunctivitis during non-pollen season. Moreover, the observed association between PM2.5 and allergic conjunctivitis has a broad public health impact on allergic diseases. Commercial Relationships: Tatsuya Mimura, None; Masao Matsubara, None Support: a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Program Number: 2488 Poster Board Number: C0008 Presentation Time: 3:45 PM–5:30 PM Phase 3 Study of Efficacy and Safety of Once-daily Olopatadine Hydrochloride, 0.77% Ophthalmic Formulation in Patients With Allergic Conjunctivitis Using the Ora Conjunctival Allergen Challenge Model (Ora-CAC™*) (NCT01743027) Eugene McLaurin1, Abhijit Narvekar2, Paul J. Gomes3. 1Total Eye Care, P.A., Memphis, TN; 2Alcon Research Ltd, Fort Worth, TX; 3VP Allergy, Ora Inc., Andover, MA. Purpose: To assess the efficacy and safety of a new, once-daily olopatadine 0.77% formulation compared with olopatadine 0.2%, olopatadine 0.1%, or vehicle for the treatment of allergic conjunctivitis. Methods: This multicenter, double-blind, parallel-group, vehicleand active-controlled study randomized patients with a history of allergic conjunctivitis (N=345; ≥18 years) 2:2:2:1 to olopatadine 0.77% (n=98), olopatadine 0.2% (n=99), olopatadine 0.1% (n=99), or vehicle (n=49). The primary endpoint was ocular itching (OI; at 3, 5 and 7 minutes post-CAC) at onset of action and 24-hours duration of action for olopatadine 0.77% versus vehicle, and OI at 24-hours ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts duration of action for olopatadine 0.77% versus olopatadine 0.2% or olopatadine 0.1%. Results: Olopatadine 0.77% was superior (p<0.0001) to vehicle for OI at all post-CAC time-points at onset and 24-hours duration of action. Differences in means (olopatadine 0.77% – vehicle) were: –1.53, –1.46, –1.17 (onset) and –1.29, –1.15, –0.89 (24 hours) for olopatadine 0.77% at 3, 5 and 7 minutes post-CAC, respectively. Olopatadine 0.77% was also superior to olopatadine 0.1% for the treatment of OI at 24-hours duration of action at all post-CAC time points, and superior to olopatadine 0.2% at 3 and 5 minutes postCAC (Table 1). There were no clinically relevant changes in safety parameters from baseline across the treatment groups, and a review of adverse events did not reveal any safety issues for olopatadine 0.77%. Conclusions: Olopatadine 0.77% was superior to vehicle, olopatadine 0.1% and olopatadine 0.2% with respect to reduction of OI associated with allergic conjunctivitis 24 hours after dosing. Olopatadine 0.77% had a safety profile comparable to olopatadine 0.1% and olopatadine 0.2%. The superior efficacy at 24 hours of olopatadine 0.77% supports potential once-daily administration and a longer duration of symptom relief compared with olopatadine 0.1% and olopatadine 0.2%. *Ora-CAC™ and Ora Calibra™ are trademarks of Ora, Inc. The Ora Conjunctival Allergen Challenge Model and Ocular Itching Scale are owned by Ora, Inc. Commercial Relationships: Eugene McLaurin, Aciex (F), Acucela (F), Alcon (F), Allergan (F), AstraZeneca (F), Bausch & Lomb (F), Inotek Pharma (F), InSite Vision (F), Lexicon Pharma (F); Abhijit Narvekar, Alcon Research Ltd (E); Paul J. Gomes, Ora, Inc. (E) Support: Alcon Research Clinical Trial: NCT01743027 Program Number: 2489 Poster Board Number: C0009 Presentation Time: 3:45 PM–5:30 PM Frequency and severity of ocular allergy symptoms in Sydney across seasons and their association with pollen levels Sailesh Kolanu1, Blanka Golebiowski1, Connie Katelaris2, Pamela Burton2, Isabelle Jalbert1. 1School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW, Australia; 2 Campbelltown Hospital, University of Western Sydney, Sydney, NSW, Australia. Purpose: Climate change can lead to an increase in levels and allergenicity of environmental pollen, posing a risk of increase in allergies. We measured the frequency and severity of ocular symptoms and their association with pollen levels across seasons. Methods: 632 university students and staff in Sydney were invited to complete a self-administered online survey. Eye Allergy Patient Impact Questionnaire (EAPIQ) and Aston University Allergy Questionnaire (AUAQ) were completed in 2012 (spring1) and 2013 (autumn, winter and spring2). Burkard volumetric trap was used for daily grass, weed, tree and total pollen counts. Frequency, troublesomeness, severity of ocular symptoms (dryness, itchiness, need to rub, burning, stinging, redness, watering, swelling/puffiness) and AUAQ symptom severity score (0-21) were compared within (one way ANOVA) and between seasons (repeated measures ANOVA). Bonferroni corrected posthoc analysis was used. Associations between pollen counts and symptoms were examined using Spearman Correlation. Results: 53 subjects completed all phases (age: 28±11 yrs, range 19-63). Tree, weed and total pollen counts were higher in spring (37±20, 8±4, 61±26 n/m3 respectively) than in other seasons (12±16, 2±2, 19±24 n/m3 respectively) (p<0.05). Grass pollen levels remained constant in spring and autumn (13±7 n/m3) but decreased to 0 in winter (p<0.001). Severity and troublesomeness of dry eyes were higher in spring2 than in autumn (p<0.03). Severity of burning eyes and red eyes were higher in spring2 than in autumn (p<0.01). Symptom severity score was higher in spring2 than in autumn or spring1 (p<0.01). Dry eyes (61-74%) and itchy/burning eyes (50-59%) were reported as the most frequent symptoms in all phases. Frequency and troublesomeness of dry eyes were higher than that of red eyes, watery eyes or swollen/puffy eyes in all seasons (p<0.05). Symptom severity score was correlated with grass (ρ=0.32;p=0.005), weed (ρ=0.35;p=0.002), tree (ρ=0.35;p=0.002) and total (ρ=0.33;p=0.03) pollen count during autumn and only weed pollen count (ρ=0.3;p=0.01) during winter. Conclusions: Mild ocular symptoms are frequent in Sydney throughout the year and their severity was higher in spring than in autumn. Further analyses including climatic variables and subject behaviour are required in order to understand the relationship between symptoms and pollen counts. Commercial Relationships: Sailesh Kolanu, None; Blanka Golebiowski, None; Connie Katelaris, None; Pamela Burton, None; Isabelle Jalbert, None Support: FRGP grant and UIPA scholarship, UNSW Program Number: 2490 Poster Board Number: C0010 Presentation Time: 3:45 PM–5:30 PM Evaluation of the Onset and Duration of Action of Topical Cetirizine (AC-170 0. 24%) for the Prevention of Allergic Conjunctivitis Paul J. Gomes1, Yesha Raval1, Emily Schoemmell1, Donna L. Welch2. 1 Allergy, ORA, Andover, MA; 2Ora, Inc, Andover, MA. Purpose: : In this phase 3, multicenter, vehicle-controlled, doubleblinded, randomized trial we evaluated the onset and duration of ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts action of topical cetirizine 0.24% using the conjunctival allergen challenge model of allergic conjunctivitis. Methods: The trial (NCT01881113) was conducted between June and September of 2013, and enrolled a total of 101 subjects; this number constituted the intent-to-treat and safety populations, while the per-protocol population included 87 subjects. Qualified subjects exhibited reproducible allergic responses to bilateral instillation of allergen. Following randomization, they were challenged with allergen on 2 separate visits, 15 minutes or 8 hours after receiving bilateral instillation of test compound or vehicle. Primary efficacy measures were ocular itching scores (at 3, 5, and 7 minutes postchallenge) and ocular hyperemia (at 7, 15, and 20 minutes postchallenge). Secondary endpoints included ciliary and episcleral redness, chemosis, eyelid swelling, and ear/palate pruritis; each of these were scored at 7, 15, and 20 minutes post challenge. Results: For the primary endpoint of ocular itching, Cetirizine was statistically superior to vehicle for the onset of action (challenge 15 minutes post-instillation; p < 0.0001) at all measured time points; the drug was also statistically superior to vehicle 8 hours after instillation (p < 0.0001). In a subgroup analysis, the drug exhibited highest efficacy in those subjects with the most severe baseline itch scores. Cetirizine was superior to vehicle for all secondary endpoints, including all measures of hyperemia. Of particular note, drug treatments were statistically superior to vehicle for prevention of eyelid swelling and ear/palate pruritis at all measured time points (p< 0.001), suggesting a unique spectrum of efficacy. The study drug was safe and well tolerated; no subjects withdrew from the study due to any adverse event. Conclusions: Cetirizine is one of the most commonly used oral agents for treatment of allergic rhinitis, but its re-formulation for topical use in treatment of allergic conjunctivitis is novel. This study demonstrates the efficacy of the drug for prevention of ocular itching associated with allergic conjunctivitis, and also establishes its unique spectrum of action against other allergic symptoms. Commercial Relationships: Paul J. Gomes, Ora, Inc (E); Yesha Raval, Ora, Inc (E); Emily Schoemmell, Ora, Inc (E); Donna L. Welch, Ora, Inc (E) Support: Supported by Aciex Therapeutics, Inc. Clinical Trial: NCT01881113 Program Number: 2491 Poster Board Number: C0011 Presentation Time: 3:45 PM–5:30 PM Allergic conjunctivitis: a national cross-sectional study Angela Castegnaro1, Federico Piliego1, Daniela Lazzarini1, Alvise La Gloria Valerio1, Paolo Mattana2, Iva A. Fregona1, Andrea Leonardi1. 1 Neuroscience, Ophthalmology Unit, University of Padua, Padova, Italy; 2Medical Service, Alfa Wassermann, Bologna, Italy. Purpose: Ocular allergy is one of the most common ocular problems in daily practice affecting 20% of the general population. A crosssectional study was conducted during the spring-summer of 2012 to evaluate clinical aspects and therapeutic approach of ocular allergy in Italy. Methods: A total of 3685 patients affected by ocular allergy were enrolled by 304 ophthalmology centers across different geographic regions in Italy. A structured questionnaire was administered to record demographic data, family history, co-morbidities, allergy test results, trigger factors, number of episodes of ocular allergy in the last year. Nine signs and symptoms were scored according the severity, frequency and duration. Patients were divide into 6 clinical forms: SAC, PAC, VKC, AKC, GPC and contact blepharoconjunctivitis (CBC). Medical treatment in the last year was recorded by drug classes. Results: Out of 3685 records, 3545 were assessable. The mean age of enrolled patients was 38±19 years (56% female). SAC (55% of patients) and AKC (7%) were equally distributed among the different age groups, while PAC (18%) increased with age and VKC (8%) was more frequent under age of 16. GPC (4%) was more frequent in the group 16-30 year of age. CBC (7%) was more frequent over 45. In the entire population of patients, itching and redness were reported in 94% and 85%, respectively, lid skin involvement in 22% of cases and keratitis in 11%. However, only 35% of patients underwent to an allergy diagnostic evaluation, with positive results in 40% of cases. 68% of the patients reported 1-5 episodes of conjunctivitis in the last year, 6% >10 episodes/year. Considering the entire patients population, over the counter decongestants/ antihistamines were used in 43%, corticosteroids in 41%, topical antihistamines in 29%, systemic antihistamines in 27%, mast cell stabilizers in 15% and antibiotics in 6% of cases. In the SAC group, 26% used systemic antihistamines, 31% topical antihistamines and 44% over the counter decongestants/antihistamines. Corticosteroids were used in 67% of GPC, 55% of VKC, 53% of AKC, 47% of CBC, in 43% of PAC and 28% of SAC. Conclusions: This survey, based on a simple questionnaire, has provided extremely useful information regarding clinical characteristics, risk factors and treatment options of a large cohort of patients affected by different forms of ocular allergy and stimulates further studies on the clinical evolution and management of these diseases. Commercial Relationships: Angela Castegnaro, None; Federico Piliego, None; Daniela Lazzarini, None; Alvise La Gloria Valerio, None; Paolo Mattana, None; Iva A. Fregona, None; Andrea Leonardi, None Program Number: 2492 Poster Board Number: C0012 Presentation Time: 3:45 PM–5:30 PM Retrospective study to evaluate the efficacy on vernal keratoconjunctivitis (VKC) of 2% Ectoine versus 0.05% ketotifen eye-drops Pia Allegri1, Giuseppina Marrazzo2, Chiara Ciurlo1, Antonio Mastromarino1, Silvia Autuori1, Ugo Murialdo1. 1Eye inflammatory department, Allergic Conjunctivitis Outpatients Department of Rapallo Hospital, Genoa, Italy; 2R&D Alfa Intes, Casoria, Italy. Purpose: Ectoine is a strong water structure-forming solute. It exerts cell-protective, anti-inflammatory and anti-allergic activity. VKC is a rare, severe, challenging seasonal eye-allergic childhood disease managed in our referral hospital center. We underwent a retrospective case series review to evaluate and compare, when administered in pre-allergic and allergic period, the efficacy of this solution versus 0.05% Ketotifen eye drops (Ketoftil®) on signs and symptoms of VKC Methods: We evaluated records from 64 male pediatric subjects (mean age 8.5 y ± 2 months) and divided it into two groups of patients treated three times a-day with Ectoine 2% (Group A) and patients treated with 0.05% Ketotifen eye drops (Group B). The included patients were evaluated from February 2013 until November 2013 on three visits (at the beginning of treatment, 3 (±10 days) months later and 6 (±20 days) months).Main criteria of statistical evaluation were for VKC slit-lamp signs: Focal or diffuse conjunctival hyperemia; BUT; Modified Oxford scale; VKC grading (modified Bonini scale); and for VKC signs: VAS scale grading ( ocular pain, itching, tearing, photophobia and foreign body sensation); Quick questionnaire on tolerance of the formulation. We compared the previous year treatment and evaluated the beginning of Cyclosporine eye drops treatment compared to the previous season ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts and the different Cyclosporine dosages (0.25%-0.5%-1%-2%) used before Results: When evaluating symptoms and signs of VKC patients, both groups showed a significant improvement (p<0.0001) from baseline and at three and six months after treatment. Althought we found a significant difference (p<0.0001) between the two treatments on the tolerability at each time point. Furthermore both drugs delayed the beginning of treatment with Cyclosporine eye drops and reduced the concentration dosage. Conclusions: Our case series review allowed us to establish that both topic treatments with 2% Ectoine as well as 0.05% Ketotifen are effective in improving signs and symptoms of VKC and to delay the adjuvant treatment with Cyclosporin. Therefore, we can conclude that Ectoine (a natural compound without any side effects) can be considered, in efficacy, equal to Ketotifen and is better tolerated by pediatric patients. Further studies with the inclusion of a bigger number of subjects are required. Commercial Relationships: Pia Allegri, None; Giuseppina Marrazzo, Alfa Intes (E); Chiara Ciurlo, None; Antonio Mastromarino, None; Silvia Autuori, None; Ugo Murialdo, None Program Number: 2493 Poster Board Number: C0013 Presentation Time: 3:45 PM–5:30 PM Suppression of experimental autoimmune uveitis (EAU) and recovery from uveitis correlate with expansion of regulatory B cells (Breg) Chengrong Yu, Ivy M. Dambuza, Sung-Hye Kim, Rashid M. Mahdi, Charles Egwuagu. Laboratory Immunology, National Eye Inst/NIH, Bethesda, MD. Purpose: Regulatory B cells (Bregs), characterized by expression of the immunoregulatory cytokine IL-10, have been implicated in the suppression of excessive inflammatory responses that lead to inflammatory and autoimmune mediated diseases (Ann Rev Imm 30:221, 2012). However, it is not clear whether it has a role in suppressing intraocular inflammation and/or the maintenance of ocular immune privilege. In this study, we have therefore examined whether the suppression of experimental autoimmune uveitis (EAU) or recovery from EAU correlates with expansion of Bregs. Methods: Autoimmune uveitis was induced by active immunization with IRBP (interphotoreceptor retinoid-binding protein in CFA (Complete Freund’s adjuvant) in C57BL/6J mice. Disease progression was monitored by clinical grading of EAU by fundoscopy and histology. To characterize B cell populations recruited into the spleen and draining lymph-nodes at the peak of the disease (day 21 post-immunization) or during the recovery phase of EAU (day 28 post-immunization) single cell suspensions from these tissues were stained with anti-CD3, CB4, CD19, CD1d, CD5, CD21, CD23, IgM, and IgD and analyzed by multicolor flow cytometry. Cells were also stimulated with LPS/PMA/Ionomycin for 5 hour and intracellular IL-10 was measured FACS. Naïve CD19+ B cells were sorted and then activated by anti-CD40 antibody or LPS for IL-10 detection by RT-PCR and intracellular cytokine staining. Results: We show that total number of B cells were significantly increased in spleen and draining lymph nodes during EAU. We further show that the major regulatory B cell population at day 21 post immunization were CD1dhi CD5hi IL-10-producing B cells. Interestingly, we found that there were twice as many IL10-producing B cells (5.4%) than IL-10-producing T cells (2.4%), suggesting that Breg may play a role during the recovery phase of the disease. Conclusions: We show here for the first time that Breg are expanded during autoimmune uveitis and may play important role in suppressing intraocular inflammation and in the maintenance of ocular immune privilege. Commercial Relationships: Chengrong Yu, None; Ivy M. Dambuza, None; Sung-Hye Kim, None; Rashid M. Mahdi, None; Charles Egwuagu, None Program Number: 2494 Poster Board Number: C0014 Presentation Time: 3:45 PM–5:30 PM Immunosuppressive function of exosomes isolated from circulating blood of the mice with experimental autoimmune uveitis (EAU) Guomin Jiang, Amir R. Hajrasouliha, Yunsong Wang, Henry J. Kaplan, Hui Shao. Department of Ophthalmology, University of Louisville, Louisville, KY. Purpose: To determine whether exosomes isolated from the sera of EAU mice contain uveitogenic interphotoreceptor petinoid binding protein (IRBP), and whether these IRBP-containing exosomes are immunogenic or tolerogenic. Methods: EAU was induced in C57BL/6 (B6) mice by immunization with IRBP1-20 peptides emulsified with complete Freund’s adjuvant. Exosomes were extracted from the sera of naïve and diseased mice by ultra-speed centrifuge. The presence of IRBP in the exosomes was examined by western blot using anti-IRBP Ab. IRBP-specific T cell responses after incubation with IRBP containing exosomes were tested by T cell proliferation assay. The severity of EAU treated with or without IRBP-containing exosomes was evaluated by histology. Results: Exosomes from the sera of EAU mice expressed significantly high level of IRBP compared to those from naïve mice. These IRBP containing exosomes inhibited the proliferation of uveitogenic T cells in vitro and suppressed the uveitis induced by IRBP-specific T cells in vivo. Conclusions: IRBP-containing exosomes was detected in the peripheral blood, which might deliver immunosuppressive signals to autoimmune T cells and might be used as a novel therapy for autoimmune uveitis. Commercial Relationships: Guomin Jiang, None; Amir R. Hajrasouliha, None; Yunsong Wang, None; Henry J. Kaplan, None; Hui Shao, None Support: NEI/NIH grant EY12974 (HS), RPB Lew R Wasserman Merit Award (HS), Commonwealth of Kentucky Research Challenge Trust Fund (HK). IRIG grant of university of Louisville (GJ). Fight for Sight (GJ) Program Number: 2495 Poster Board Number: C0015 Presentation Time: 3:45 PM–5:30 PM Intravitreal leukocytes change plasma cell membrane expression pattern in spontaneous recurrent uveitis Cornelia A. Deeg1, Nina B. Burkhardt1, Roxane L. Degroote1, Barbara Amann1, Marius Ueffing2, 3, Stefanie M. Hauck2. 1Department of Veterinary Sciences, LMU Munich, Munich, Germany; 2Helmholtz Center Munich, Research Unit for Protein Scienceute for Animal Physiology, Munich, Germany; 3Centre for Ophthalmology, University of Tübingen, Institute for Ophthalmic Research, Tübingen, Germany. Purpose: Horses frequently (10%) develop spontaneous recurrent uveitis (ERU), an autoimmune mediated disease. Pathophysiology is driven by lymphocytes that enter the inner eye through outer bloodretinal barrier and destroy retinal proteins. In order to understand differences in cell surface phenotype of peripheral blood derived lymphocytes and intraocular lymphocytes, we chose a differential proteomics approach, determining plasma cell surface protein phenotypes in healthy and diseased cells. ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Methods: Peripheral blood derived lymphocytes (PBL) were separated from horses with spontaneous equine recurrent uveitis, that were presented to Equine Clinic of LMU Munich for therapeutic vitrectomy. Intraocular lymphocytes (VL) were separated from vitreous obtained during pars plana surgery. Amount of PBL used in experiments was adjusted to total amount of cells obtained from intraocular surgery (around 1 x 104 cells) for comparison of protein expression. Cell surface proteins were covalently labeled with biotin, affinity purified and released by PNGase F. Detection, identification and quantification of respective proteins was carried out with label free mass spectrometry. Results: Our approach resulted in clear identification of 206 proteins in PBL and 212 proteins in VL preparation of plasma cell membrane proteins. Of these, 196 proteins were identified in both fractions. Overall, eight proteins were ≥ 5 fold enriched in immune cells separated from vitreous. In contrast, 14 proteins were enriched in PBL fraction (factor ≥ 5). Interestingly, upregulated proteins in VL comprise activated sushi domain containing 2, matrix metallopeptidase 25 and leukocyte cell adhesion molecule. Downregulated candidates were semaphorin 4D, atlastin GTPase 3, interferon (alpha, beta and omega) receptor 1 and Fas. Conclusions: Cell surface analysis of ERU effector cells was suited to generate a cell surface proteome profile and to detect specific changes in intraocular lymphocytes of ERU cases. This will help to understand inflammation associated changes in a spontaneous autoimmune disease. Commercial Relationships: Cornelia A. Deeg, None; Nina B. Burkhardt, None; Roxane L. Degroote, None; Barbara Amann, None; Marius Ueffing, None; Stefanie M. Hauck, None Support: DFG DE 719/4-1 Program Number: 2496 Poster Board Number: C0016 Presentation Time: 3:45 PM–5:30 PM Complete Freunds Adjuvant can protect from Retinal Autoimmunity by an Interleukin-10 dependent mechanism Rafael S. Grajewski, Rebecca Scholz, Deniz Hos, Claus Cursiefen, Ludwig M. Heindl. Department of Ophthalmology, University of Cologne, Cologne, Germany. Purpose: Complete Freunds Adjuvant (CFA) is an oily emulsion with mycobacterial components that is used as an adjuvant to induce antigen-specific autoimmunity, such as experimental autoimmune uveitis (EAU). Our previous work demonstrated that CFA is also capable of non-specific activation of regulatory T cells (Treg). The purpose of the present study was to analyze the immunoregulatory properties of CFA in EAU. Methods: Wild-type (WT) and IL-10 knockout (KO) C57Bl/6 mice were pre-treated by subcutaneous injection of CFA or Incomplete Freunds Adjuvant (IFA) seven days before induction of EAU by immunization with IRBP peptide 1-20 emulsified in CFA and with Pertussis-Toxin (PTX). EAU scores (scale ranging in severity from 0= no disease to 4 = complete retinal destruction) and associated immunological responses (delayed type hypersensitivity, DTH) were examined. Results: Pre-treatment with CFA in WT mice (CFA/WT) significantly reduced incidence (p=0.049) and severity (p=0.005) of EAU compared to IFA pre-treatment (IFA/WT). Conversely, pre-treatment of KO mice with CFA (CFA/KO) did not alter incidence and severity of EAU, when compared to IFA pre-treatment of KO mice (IFA/ KO). Comparably, the DTH response was significantly lower in the CFA/WT than in the IFA/WT group (p=0.016) and not significantly different in the CFA/KO versus the IFA/KO group. EAU scores and incidence, as well as DTH were not statistically different between the control groups of the two mouse strains (IFA/WT versus IFA/KO). Conclusions: Although CFA is needed to induce EAU, it partially also has immunosuppressive properties that may be utilized, when it is administered before immunization with antigen. This effect seems to represent an active immunosuppressive mechanism rather than being due to passive exhaustion of effector mechanisms, because it is dependent on the presence of IL-10. Commercial Relationships: Rafael S. Grajewski, None; Rebecca Scholz, None; Deniz Hos, None; Claus Cursiefen, None; Ludwig M. Heindl, None Support: German Research Foundation: DFG GR 2647/4-1 Program Number: 2497 Poster Board Number: C0017 Presentation Time: 3:45 PM–5:30 PM Altering the gut microbiota ameliorates experimental autoimmune uveitis Yukiko Nakamura1, Christina Metea1, Henry Gruner1, Mark Asquith3, Stephen R. Planck1, 2, James T. Rosenbaum1, 2, Phoebe Lin1. 1Casey Eye Institute, Oregon Health & Science University, Portland, OR; 2 Ophthalmology, Devers Eye Institute, Portland, Oregon, Portland, OR; 3Rheumatology, Oregon Health and Science University, Portland, OR. Purpose: The gut microbiota appear to be important in the regulation of a number of immune-mediated diseases including multiple sclerosis, diabetes mellitus, and inflammatory bowel disease, but the relationship between the gut microbiome and autoimmune uveitis has not yet been determined. The objective of this study was to investigate whether altering the gut microbiota with broad-spectrum antibiotics affects the severity of experimental autoimmune uveitis (EAU). Methods: Interphotoreceptor binding protein (IRBP) peptide 161-180 was used to induce uveitis in B10.RIII mice. Mice were treated with oral or intraperitoneal (ip) antibiotics (1 mg/mL each of ampicillin, neomycin sulfate, and metronidazole, 0.5 mg/mL of vancomycin) starting 1 week prior to antigen challenge. Eyes, spleen, cervical and mesenteric lymph nodes (CLN, MLN), and lamina propia lymphocytes (LPL) were collected on day 20-21. We performed clinical and histological grading of eyes, as well as flow cytometry analysis for quantifying regulatory T-cell (Treg) populations. Results: A smaller proportion of antibiotic-fed animals had clinical EAU scores ≥ 2.5 compared with water-fed animals (7.7% vs 46.1%, p=0.048, OR=9.0) (Fig. 1a). The histopathological EAU scores followed a similar trend with 22.2% eyes in the antibiotic-fed group compared to 76.9% eyes in the water-fed group developing a score ≥ 2.5 (p=0.011, OR=11.67, Fig. 1b). No significant difference in scores was found between ip antibiotic compared to ip control groups (clinical: 33% vs. 44.4%, p=0.625, OR=0.629; histopathological: 50% vs 75%, p=0.302, OR=0.33). There were higher proportions of Treg (CD4+, FoxP3+) cells in the CLN, MLN, spleen, and LPL of oral antibiotic-fed mice compared to water-fed mice. Conclusions: Our results suggest that alteration of the gut microbiota by oral antibiotic administration modulates the development of EAU. Further investigation of the Treg populations associated with the gut microbial change will be warranted to determine the underlying mechanism. ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Commercial Relationships: Gerhild Wildner, None; Andrea Huber, None; Maria Diedrichs-Möhring, None Fig. 1. Altering the gut microbiota ameliorates EAU in B10.RIII mice.(A) Clinical scores at peak inflammation (day 13-14) in 3 experiments (n=4-5, each experiment)(B) Histological scores at the end of the experiment (day 20-21) in 2 experiments (n=4-5, each experiment). Commercial Relationships: Yukiko Nakamura, None; Christina Metea, None; Henry Gruner, None; Mark Asquith, None; Stephen R. Planck, None; James T. Rosenbaum, None; Phoebe Lin, None Program Number: 2498 Poster Board Number: C0018 Presentation Time: 3:45 PM–5:30 PM Therapy of relapsing-remitting experimental uveitis in rats by oral tolerance induction Gerhild Wildner, Andrea Huber, Maria Diedrichs-Möhring. Ophthalmology, Clinic of the University of Munich LMU, Munich, Germany. Purpose: Antigen-specific tolerance induction would be a desired therapy for uveitis patients. Our relapsing-remitting EAU rat model, induced by immunization with IRBP peptide R14 offers the possibility to test the effect of oral tolerance induction on the prevention of relapses. We tested several peptides with sequences overlapping the amino acid sequence of R14 for preventing uveitis, and different doses of R14 for therapy. High dose of oral antigen should cause clonal deletion, low dose induce regulatory T cells, thus a combination of both, deleting autoreactive T cells and induce Tregs should be most effective. Methods: Lewis rats were immunized with R14-CFA to induce EAU. Oral tolerance was induced prior to immunization (prophylaxis) with 200mg peptide 3x every other day before immunization, or from onset of EAU to termination to prevent relapses (therapy). For prophylaxis we also fed peptides overlapping the sequence of R14. Therapeutic feeding was performed with 200mg R14 during EAU, 3x2mg R14 followed by 200mg or 3x2mg R14 only after onset of EAU. Control groups received PBS only. Mesenteric LN cells were tested for surface markers, cytokines and Foxp3 expression. Results: Only preventive feeding of R14 and R16, but not of overlapping peptides suppressed EAU. R14 was more tolerogenic than R16 and also prevented relapses (EAU score PBS group: 2.4; R14-fed: 0.56; R16-fed: 1.25). Therapeutic feeding with R14 had no effect on the primary course of EAU, but reduced relapses to 19% of eyes and 23% of rats compared to 50% and 69% relapses in the control group). High-dose feeding (2mg) only had no effect, and 2mg+200mg feeding was less effective (23% relapses (eyes) vs. 31% of rats) than low-dose feeding only. After in vitro-stimulation with R14 TCRab+/CD4+ T cells increased in all R14-fed groups, while TCRab+/CD4+/CD8+ cells and IL-10-producing TCRab and TCRgd cells decreased. Less than 1% of the mLN T cells produced IL-17 or IFN-g, and less than 3% IL-10. Foxp3-expressing TCRab cells slightly increased to 1.4% in the 200mg fed group only. Conclusions: Therapeutic feeding of R14 was highly effective in preventing relapses, making oral tolerance a useful therapy for relapsing-remitting uveitis. High- and low dose combination did not improve tolerance. There was no clear association with any of the analyzed mLN populations and successful oral tolerance induction. Program Number: 2499 Poster Board Number: C0019 Presentation Time: 3:45 PM–5:30 PM Spontaneous ocular autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen Reiko Horai, Phyllis B. Silver, Jun Chen, Carlos Zárate-Bladés, Wai Po Chong, Ru Zhou, Yingyos Jittayasothorn, Sonia Nguyen, ChiChao Chan, Rachel R. Caspi. Laboratory of Immunology, NEI, NIH, Bethesda, MD. Purpose: The EAU model, induced by immunization of mice with the retinal protein IRBP or its peptides, has been very useful to study basic mechanisms of ocular inflammation. However, it is inadequate for some types of studies, due to the need for active immunization in the context of strong bacterial adjuvants. To understand the pathogenesis of uveitis under more physiological settings, we generated transgenic mice that express a retina-specific T cell receptor (TCR). Methods: Transgenic constructs incorporating the IRBP161-180specific TCR α and β chains under CD2 or MHC class I promoters, respectively, were co-injected into embryos of EAU-susceptible B10. RIII mice. IRBP-specific T cells were detected with an Ag-MHC class II-Ig dimer. Ocular pathology was evaluated by fundoscopy and histology. Results: Three lines of IRBP TCR Tg mice (R161H, M, L) were established that express different levels of the transgenic TCR and show different proportions of IRBP-specific CD4+ T cells in their peripheral repertoire. Importantly, two of the lines, R161H and R161M, rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third line, R161L, appeared “poised” and only developed appreciable disease upon immune perturbation by microbial stimuli. Susceptibility roughly paralleled expression of the transgenic TCR. Peripheral IRBP-specific CD4+ T cells displayed a naïve phenotype, whereas T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory (Treg) cells. Retina-specific Treg cells appeared to have been peripherally converted rather than thymically derived. T cells from R161 mice responded to IRBP and transferred uveitis to naïve recipients, providing a source of retina-specific T cells for a variety of basic studies. Examples that will be presented include the role of commensal microbiota in uveitogenic T cell priming, as well as effector cytokines and regulatory mechanisms in uveitis. Conclusions: R161 lines of transgenic mice provide a new and valuable model of spontaneous autoimmune uveitis that allows to study natural triggers and basic mechanisms involved in breakdown of immune homeostasis in the eye. This model may also be useful for the development of therapeutic strategies for uveitis. Commercial Relationships: Reiko Horai, None; Phyllis B. Silver, None; Jun Chen, None; Carlos Zárate-Bladés, None; Wai Po Chong, None; Ru Zhou, None; Yingyos Jittayasothorn, None; Sonia Nguyen, None; Chi-Chao Chan, None; Rachel R. Caspi, None ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Program Number: 2500 Poster Board Number: C0020 Presentation Time: 3:45 PM–5:30 PM Characterising the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in experimental autoimmune uveoretinitis (EAU) Gemma Beers1, Joanne Boldison2, David A. Copland1, Peter S. Adamson3, Lindsay B. Nicholson1, 2, Andrew D. Dick1, 2. 1Academic unit of Ophthalmology, University of Bristol, Bristol, United Kingdom; 2School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom; 3Ophthalmology Discovery Performance Unit, GlaxoSmithKline, Stevenage, United Kingdom. Purpose: Macrophage activation can be regulated via hydrolysis of oxidised low density lipoproteins (oxLDL) by Lp-PLA2. This produces lysophosphatidylcholine and non-esterified fatty acids that up-regulate expression of chemokines and adhesion molecules, induce macrophage migration and promote pro-inflammatory cytokine release. Inhibition of this enzyme may therefore perturb macrophage function and attenuate inflammation. We utilised Lp-PLA2 knockout (KO) mice to determine the effect of LpPLA2 depletion in autoimmune retinal inflammation, employing a murine model of autoimmune uveitis, EAU, in which macrophages are central to progression and expression of disease. Uveitis is a prominent cause of visual impairment, found commonly in the working age population. As such, research into the underlying mechanisms of the disease and development of new treatment options is important to overcome the medical, social and financial implications associated with this pathology. Methods: C57BL/6 Lp-PLA2 KO mice, heterozygotes (HET) and wild type (WT) controls were immunized to induce EAU by subcutaneous injection of RBP-3 (IRBP)1-20 peptide in Complete Freund’s adjuvant, plus intra-peritoneal (IP) injection of heat inactivated pertussis toxin. Clinical disease was monitored by Topical Endoscopic Fundus imaging (TEFI). Mice were sacrificed on day 26 post immunisation, infiltrating leukocytes were quantified by flow cytometry and histological disease severity was assessed. Results: Lp-PLA2 KO mice showed substantially fewer infiltrating CD45+ cells compared to both WT and HET controls, which correlated with a lower disease score by TEFI and histology. In addition to a reduction in the absolute number of infiltrating cells in the retina, KO mice also exhibited an altered ratio of leukocyte populations compared to HET and WT controls. An apparent reduction in the number of CD4+ infiltrating cells in Lp-PLA2 KO retina may indicate an effect on T cell priming and subsequent tissue infiltration in these animals. Conclusions: Lp-PLA2 KO mice, immunised to induce EAU experienced decreased macrophage infiltration and less clinical disease. The observation of suppressed clinical and histological disease score and number of infiltrating cells infers a reduction in both activation and migration of inflammatory cells via depletion of Lp-PLA2. Commercial Relationships: Gemma Beers, None; Joanne Boldison, None; David A. Copland, None; Peter S. Adamson, GlaxoSmithKline (E); Lindsay B. Nicholson, None; Andrew D. Dick, None Support: Biotechnology and Biological Sciences Research Council (BBSRC) and GlaxoSmithKline (GSK) Program Number: 2501 Poster Board Number: C0021 Presentation Time: 3:45 PM–5:30 PM P2X7 Deficiency Protects Against Experimental Autoimmune Uveitis Simon R. Taylor1, 2, Shenzhen Tempest-Roe1, Emily Shao1, 2, John McDaid1. 1Immunology & Inflammation, Imperial College London, London, United Kingdom; 2Ophthalmology, Royal Surrey County Hospital, Guildford, United Kingdom. Purpose: The immune system is designed to discriminate between self and non-self, but also to detect foreign antigens in the context of ‘danger’. It has been suggested that ATP provides such a stimulus, acting via the purinergic P2X7 receptor, and that pathological stimulation of the P2X7 receptor may be involved in the development of autoimmune disease. We therefore explored the impact of P2X7 deficiency on the development of Experimental Autoimmune Uveitis (EAU) in mice, to whether this receptor may be a viable therapeutic target. Methods: EAU was induced in two strains of P2X7-/- mice (which were both generated on a C57BL/6 background) and in C57BL/6 control animals, using 500 mg IRBP peptide 1-20 (GPTHLFQPSLVLDMAKVLLD) in PBS emulsified in CFA. Additional adjuvant of 1.5 mg Bordetella pertussis toxin was injected into the peritoneal cavity. Procedures were performed under a Home Office License in accordance with the regulations of the United Kingdom Animal (Scientific Procedures) Act (1986). Animals were monitored using topical endoscopic fundal imaging and culled at day 28, when histological examination of eyes was undertaken. Results: P2X7 deficiency protected against the development of EAU, with disease scores being significantly lower in both strains of genetargeted animals compared to control animals (Figure 1). There were also differences in disease scores between the two strains of genetargeted animals, with Pfizer P2X7-/- showing lower disease activity scores than GlaxoSmithKline P2X7-/- animals. Conclusions: P2X7 deficiency protects against the development of EAU in mice and may represent a viable therapeutic target for ocular inflammatory disease. Intra-strain differences may reflect the differences in gene targeting approaches and selective deletion (or failure of deletion) of splice variants. Figure 1: Representative images taken at day 21 post-inducationof EAU in wild-type C57BL/6 animals (A,B); GSK P2X7ko animals (C,D); Pfizer P2X7ko animals (E,F). Commercial Relationships: Simon R. Taylor, None; Shenzhen Tempest-Roe, GlaxoSmithKline (F); Emily Shao, None; John McDaid, None Support: NIHR CDF-2011-04-051; BBSRC BB/K501037/1 ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Program Number: 2502 Poster Board Number: C0022 Presentation Time: 3:45 PM–5:30 PM A role for the Syk-CARD9 pathway in the pathogenesis of autoimmune uveitis Ellen J. Lee1, 3, Brieanna Brown3, 1, Emily Vance3, 1, Maya Lewinsohn3, Phyllis B. Silver4, Rachel R. Caspi4, Holly L. Rosenzweig2, 1. 1 Ophthalmology, Oregon Health & Science University, Portland, OR; 2 Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR; 3Veterans Affairs Medical Center, Portland, OR; 4Laboratory of Immunology, NEI/NIH, Bethesda, MD. Purpose: While the role of adaptive T cell responses in uveitis has been extensively studied, we know little of the innate signals that precede and contribute to autoreactive T cell activation and disease. We, therefore, investigated the C-type lectin receptors (CLRs) in the T cell-dependent uveitis model, experimental autoimmune uveitis (EAU). Methods: EAU was induced in Card9 knockout (KO) mice and C57BL/6J congenic wild-type (WT) controls by immunization with interphotoreceptor retinoid-binding protein (IRBP). Pathway-focused gene expression changes were quantified by real-time PCR-based multiplex arrays at d10 post-immunization. At d21, uveitis was assessed by fundus imaging and histopathology. Flow cytometry was used to quantify cellular infiltrate of eyes and antigen-specific CD4+ T cell effector responses from IRBP-stimulated splenocytes. The Syk inhibitor, piceatannol (5mg/kg x 4 i.p. injections), versus vehicle control was evaluated in WT mice (n=6/treatment). Results: Multiplex array analysis of isolated neuroretina revealed early induction of genes involved in fungal and mycobacterial host defense responses, including Dectin-1, Dectin-2, and Mincle, which comprise a subgroup of CLRs that transduce signals through Syk and CARD9. Card9 expression was essential for induction of “fungalassociated” transcriptional response and contributed functionally to EAU, as CARD9 KO mice were resistant to EAU. Card9 deficiency significantly impaired EAU development, as assessed by clinical fundus and histopathologic grading (WT=2.5±0.34 vs. KO=0.28±0.11, p<0.001), as well as by reduced leukocyte infiltration into the vitreous (WT=159±41 vs. KO=4±2, p<0.0001). Flow cytometry of whole eyes revealed marked reduction of CD4+ T cells, neutrophils and monocyte-macrophages. Card9 expression was essential for induction of IRBP-specific T cell effector responses, as KO mice demonstrated greater than 2-fold reduction in frequency of both Th1 and Th17 cells. The resistance of CARD9 KO mice to EAU was reproduced in WT mice by pharmacologic inhibition of the upstream kinase Syk, further underscoring the importance of the SykCARD9 pathway in orchestration of EAU. Conclusions: Our studies uncover a role for the Syk-CARD9 pathway in induction of uveitogenic T cell responses and autoimmune uveitis and implicate CLRs as important players in uveitis. These pathways could constitute new targets for treatment of ocular inflammatory disease. Commercial Relationships: Ellen J. Lee, None; Brieanna Brown, None; Emily Vance, None; Maya Lewinsohn, None; Phyllis B. Silver, None; Rachel R. Caspi, None; Holly L. Rosenzweig, None Support: NEI/NIH grant EY019020, Research to Prevent Blindness Foundation, NEI Intramural support EY000184-30 Program Number: 2503 Poster Board Number: C0023 Presentation Time: 3:45 PM–5:30 PM Characterization of the Role of IRF-8 in the Retina During Autoimmune Uveitis Sung-Hye Kim, Chengrong Yu, Charles Egwuagu. Lab of Immunology, NEI, Bethesda, MD. Purpose: Interferon Regulatory Factor (IRF)-8 is required for the development, maturation and expression of anti-microbial defenses of myeloid cells and regulates CFH expression in retinal microglial cells. However, IRF-8-dependent network contains several genes that enhance susceptibility to infection while others confer complete protection against parasitic diseases such as cerebral malaria. We recently showed that retinal cells constitutively express low levels of IRF-8 and its expression increased significantly in the retina during intraocular inflammation. However, the functional significance of IRF-8 expression in retina remains unclear. In this study, we have generated mice with targeted deletion of IRF-8 in the retina to investigate the function of IRF-8 during intraocular inflammation. Methods: We generated two IRF-8 conditional knockout mouse strains by breeding IRF-8 floxed mice (IRF-8 fl/fl) (kind gift from Herbert Morse, NIH) with mice expressing Cre under direction of the α-Cre promoter (α-CRE) or the Rx promoter (Rx). Experimental autoimmune uveitis (EAU) was induced by active immunization with IRBP/CFA. Disease severity was characterized by fundoscopy, optical coherence tomography (OCT), histology and FACS. Visual function was assessed by electroradiography (ERG). Results: The WT mice developed severe EAU characterized by massive infiltration of inflammatory cells into the retina, photoreceptor cell loss, focal retinitis, retinal vasculitis and multifocal choroiditis, In contrast, the two IRF-8 KO mouse strains developed very mild EAU, suggesting that IRF-8 deletion conferred protection against EAU. However, visual function tests reveal alterations in dark and light adapted ERG in both IRF-8 KO mouse strains. Conclusions: Our data confirm the pleiotropic effects of IRF-8 in different tissues and suggests that IRF-8 expression may have double-edged effects in the retina. On the one hand, IRF-8 induces transcription of CFH gene and may thus confer protection against age-related macular degeneration (AMD). On the other hand, the reduced inflammation in the IRF-8 KO mice suggest that IRF-8 may promote inflammatory responses in the retina and that therapeutic targeting of IRF-8 may be used to alleviate uveitis. Commercial Relationships: Sung-Hye Kim, None; Chengrong Yu, None; Charles Egwuagu, None Program Number: 2504 Poster Board Number: C0024 Presentation Time: 3:45 PM–5:30 PM The role of peripheral dendritic cells in immune activation in anterior uveitis Conor Murphy1, Micheal O’Rourke1, 2, Mary Connolly2, Cheryl Sweeney2, Ursula Fearon2. 1Ophthalmology, RCSI, Dublin, Ireland; 2 Department of Rheumatology, University College Dublin, Dublin, Ireland. Purpose: Pathogens, such as microbial components express pathogen associated molecular pathogens. These can interact with innate pattern recognition receptors via toll like receptors (TLRs) on antigen presenting cells (APCs). TLRs play a critical role linking innate and adaptive immunity promoting the maturation of APCs through the production of pro-inflammatory cytokines and the up-regulation of co-stimulatory molecules. This interaction also allows APCs to become efficient in the presentation of specific antigens to naïve T cells initiating adaptive immunity. It is evident that these processes can be altered by a class of small non-coding RNAs or microRNA (miR) which exert their biological function through suppression of their target genes, abnormal expression of which has been demonstrated in chronic inflammatory diseases. Methods: Peripheral blood mononuclear cells were isolated from active AU patients and healthy controls (n=5). CD14+ monocytes (purity >97%) isolated by positive selection using magnetic bead separation were differentiated into imDCs by culturing for 7 days ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts with IL-4 and GM-CSF and matured in media for 24 hours. The effects of TLR activation and pro-inflammatory stimuli were examined by culturing moDC with Pam3CSK4 (1μg/ml), Poly I:C (25μg/ml), LPS (1μg/ml), IL-1β (10ng/ml) and TNFα (10ng/ml). Cell surface expression of CD83 (maturity) and CD86 (activation) on DC were quantified by flow cytometry. Cytokine analysis was performed on supernatant by ELISA. Total RNA from the mature DC was isolated using the miRneasy isolation kit. Quantification of miR expression was analyzed by real-time PCR, using miRNA-let-7a as an endogenous control. Results: CD83 and CD86 expression on AU DC was increased in response to all agonists compared to basal, most notably for TLR3 and IL1b. A significant increase in CD83 mean fluorescent intensity (MFI) in response to IL1b was demonstrated in AU moDC compared to HC (p=0.05). This was paralled by an increase in IL12 (p=0.05) and IL23 expression in cultured supernatants. Furthermore, expression of miR155 was also increased in response to Poly I:C, LPS and Il1b in AU DC. Conclusions: These results support peripheral activation of DC by TLR3 and Il1b in AU patients suggesting a possible role in AU. Altered miR155 expression in DC from AU patients suggests that this miR may contribute to the pathogenesis of AU by mediating TLR3 activated pro-inflammatory pathways. Commercial Relationships: Conor Murphy, None; Micheal O’Rourke, None; Mary Connolly, None; Cheryl Sweeney, None; Ursula Fearon, None Program Number: 2505 Poster Board Number: C0025 Presentation Time: 3:45 PM–5:30 PM Distinct Patterns of Fundus Autofluorescence in Paraneoplastic and Non-Paraneoplastic Forms of Autoimmune Retinopathy Responding to Systemic Immunosuppressive Therapy Sarwar Zahid1, 2, Melisa Nika2, Amani Al-Tarouti2, Kari E. Branham2, John R. Heckenlively2, Thiran Jayasundera2. 1Henry Ford Hospital, Detroit, MI; 2Kellogg Eye Center, Ann Arbor, MI. Purpose: To describe distinct patterns of fundus autofluorescence (FAF) in patients with paraneoplastic (pAIR) and non-paraneoplastic autoimmune retinopathy (npAIR) who demonstrate a clinical response to systemic immunosuppression. Methods: A retrospective study was conducted of 35 patients with AIR, who met the following diagnostic criteria: 1) presented with sudden adult-onset rapidly progressive visual disturbance; 2) exhibited abnormal full-field ERG (ffERG) parameters; 3) antiretinal antibody positivity, including anti-recoverin. Clinical data focused on FAF findings in patients who exhibited objective clinical improvement after immunosuppressive treatment as detected by a 25% improvement in quantified Goldmann visual fields (GVF) or visual acuity (VA) during follow-up. Results: Twenty-four patients with npAIR and 11 patients with pAIR exhibited improvement after immunosuppressive treatment during follow-up. Sixteen patients had FAF imaging available. Fourteen of these patients exhibited a ring of parafoveal hyperfluorescence; 2 of these patients had hyperfluorescence outside the vascular arcades as well. Two patients exhibited central foveal hyperfluorescence, while 4 patients exhibited peripheral hypofluorescence outside and along the vascular arcades suggestive of retinal pigment epithelium (RPE) atrophy. Three patients exhibited peripapillary atrophy with surrounding hyperfluorescence. FAF findings were symmetric in each eye irrespective of interocular differences in GVF areas or VA in each patient. Conclusions: Patients with suspected AIR that exhibit improved visual function with treatment constitute an ideal group in which to investigate the presence of FAF signatures in this heterogeneous disease. We confirm that FAF changes seen in retinal dystrophies, such as concentric rings of parafoveal hyperfluorescence and central foveal hyperfluorescence, are also present in patients with AIR. Commercial Relationships: Sarwar Zahid, None; Melisa Nika, None; Amani Al-Tarouti, None; Kari E. Branham, None; John R. Heckenlively, None; Thiran Jayasundera, None Program Number: 2506 Poster Board Number: C0026 Presentation Time: 3:45 PM–5:30 PM Malignancy Presenting After Immunomodulatory Therapy for Presumed Non-Paraneoplastic Autoimmune Retinopathy Heena Patel, Janet L. Davis. Bascom Palmer Eye Institute/UMH, Miami, FL. Purpose: To describe 3 patients who developed malignancies during treatment with immunomodulatory therapy for presumed nonparaneoplastic autoimmune retinopathy (npAIR) despite negative oncologic work-ups prior to therapy. Methods: Review of clinical records of 3 patients with autoimmune retinopathy who developed malignancies during treatment. Results: Patients were a 73-year-old woman and two 72 and 63 year old men. Patient #1 presented with bilateral decreased vision and anti-CNS antibodies. The amplitudes of the ERG were moderately reduced in both eyes for rods and cones. MRI brain, lumbar puncture, vitreous cytology, CT scan of chest, abdomen, and pelvis, whole body gallium scan, SPECT scan, and bone marrow biopsy were negative for malignancy. She was treated with IV methylprednisolone, followed by oral prednisone. Eight months later she received 2 doses of IVIG. Biopsy of a submandibular lymph node 17 months following initial consult revealed small cell carcinoma of unknown origin. Patient #2 presented with nyctalopia and ERG showed absent rod responses, mixed rod and cone, and some slightly reduced cone response. Serum antibodies reacted with an unknown 40kDa protein. Oncologic work-up included CT chest, abdomen, and pelvis, PET scan, and dermatologic exam. Patient was treated with IVIG initially for 2 doses and 3 months later with immunosuppressants. Two months after starting azathioprine and cyclosporine, a cervical lymph node biopsy revealed large cell neuroendocrine carcinoma with an unknown primary. Patient #3 presented with and unexplained peripheral visual field defect and a nearly extinguished ERG in the right eye and markedly reduced ERG in the left eye. Anti-retinal antibodies were not detected. Initial oncologic work-up included CT chest, abdomen, and pelvis. Patient was treated with azathioprine, and 9 months later was diagnosed with colon adenocarcinoma following colonoscopy due to enlarged lymph nodes seen on PET scan. PET scan was performed after patient presented with new onset diaphragmatic paralysis. Conclusions: Suspected npAIR patients should have a thorough work-up for a malignancy prior to the initiation of immunomodulatory therapy. If treatment with an immunomodulatory agent is recommended, the patient should be informed of the inability to completely exclude occult malignancy and that treatment may unmask an underlying tumor as the immune system is suppressed. Commercial Relationships: Heena Patel, None; Janet L. Davis, None Program Number: 2507 Poster Board Number: C0027 Presentation Time: 3:45 PM–5:30 PM Tattoo-Associated Uveitis Trucian A. Ostheimer, Bryn Burkholder, Theresa G. Leung, Nicholas J. Butler, James P. Dunn, Jennifer E. Thorne. Ophthalmology, Johns Hopkins, Baltimore, MD. ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Purpose: To describe the clinical presentation of bilateral uveitis with coincident onset of raised and indurated tattooed skin in a case series of six patients. Methods: Six consecutive patients with coincident bilateral uveitis and cutaneous tattoo induration were evaluated at a tertiary ophthalmologic facility over the course of approximately one year. All subjects underwent complete ophthalmic examination and a focused systemic medical workup including serologic testing and imaging studies to rule out syphilis and sarcoidosis. Additional testing for specific uveitic etiologies was performed on a case-by-case basis. Two participants underwent biopsy of their tattoos. The patients’ clinical courses and responses to treatment over a follow-up period of one to twelve months are reported. Main outcome measures included degree of intraocular inflammation, ocular complications, visual acuity, clinically observable tattooed skin changes, and biopsy results. Results: Patient ages at the time of presentation ranged from 20 to 42 years of age. Five patients are African-American and one patient is Caucasian. Initial best-corrected visual acuity varied from 20/20 to 20/400. Four patients were diagnosed with bilateral non-granulomatous anterior uveitis; three with chronic and one with recurrent disease. The remaining two patients were diagnosed with bilateral chronic granulomatous panuveitis. The most significant ocular complications included uveitic glaucoma, iris bombe, severe cystoid macular edema, and a neurosensory retinal detachment. All patients had multiple tattoos, some of which were multicolored, but only areas of skin containing black pigment were affected. Biopsies of raised and indurated tattoos were performed in two patients and demonstrated non-caseating granulomatous inflammation surrounding tattoo ink in the dermis. The skin changes resolved in all patients, with a faster response noted in those treated with highdose oral prednisone for intraocular inflammation. Three patients subsequently experienced recurrent flares of intraocular inflammation in conjunction with the recurrence of raised and indurated tattoos. Conclusions: These cases may represent a subset of patients with previously undiagnosed sarcoidosis, in whom tattooing may have incited a simultaneous inflammation of the eyes and tattooed skin. Commercial Relationships: Trucian A. Ostheimer, None; Bryn Burkholder, None; Theresa G. Leung, None; Nicholas J. Butler, None; James P. Dunn, None; Jennifer E. Thorne, AbbVie (C), Allergan (F), Gilead (C), NEI (F), NIAID (F), RPB (F), Xoma (C) Program Number: 2508 Poster Board Number: C0028 Presentation Time: 3:45 PM–5:30 PM Cataract surgery with or without primary lens implantation in children with chronic uveitis Damien Guindolet1, 2, Pascal Dureau2, Catherine Edelson2, Amandine Barjol2, Céline Terrada3, Georges Caputo2, Phuc Lehoang1, Bahram Bodaghi1. 1Hôpital Pitié-Salpétrière, Paris, France; 2Fondation Ophtalmologique Adolphe de Rothschild, Paris, France; 3Hôpital Saint Louis, Paris, France. Purpose: To evaluate the management of cataract in children with uveitis and compare the visual outcome with or without primary lens implantation. Methods: Retrospective study performed between 2007 and 2012 on children with chronic uveitis who underwent a cataract surgery, with primary posterior chamber IOL implantation with a foldable hydrophobic acrylic IOL or left aphakic. Results: The study included 20 eyes of 16 patients (9 Juvenile Idiopathic Arthritis). The mean age at surgery was 8.63 years (5.6813.97) in 14 implanted eyes and 6.08 years (4.11-11.28; p=0.03) in 6 eyes which remained aphakic. The mean postoperative follow-up was 36.44 months (8.72-69.57). All patients except one were treated with methotrexate. Four patients (5 eyes) in the implanted group and 2 patients (2 eyes) in the apahakic group were additionally treated with TNF-alpha blockers. In the implanted and the aphakic groups, the preoperative mean BCVA was 1.11 logMAR (0.40-2.30) and 1.78 logMAR (0.70-2.30), respectively and the postoperative mean BCVA was 0.48 logMAR (0-3; p=0.02) and 0.47 logMAR (0-2.6; p=0.047), respectively. The baseline oral corticosteroids dosage in the implanted group and in the aphakic group was 8.14mg/d (0-30) and 6.17mg/d (0-15), respectively. It was increased to 27.9mg/d (0-55) and 25.83mg/d (15-40), respectively during the perioeprative period and tapered to 7.54mg/d (0-50) and 2.08mg/d (0-2.50), respectively at the end of the follow-up. Postoperative complications in the implanted group were posterior synachiae (5), posterior capsule opacification (1), cellular proliferation (8), cells on IOL (2), Elschnig pearls (5), secondary ocular hypertension (4), macular edema (5), retinal detachment (1). In the aphakic group complications were choroidal detachment (1) and intravitreal hemorrhage (2). Conclusions: Pediatric cataract surgery in chronic uveitis with primary posterior chamber hydrophobic IOL implantation is possible and leads to a good and prompt visual rehabilitation. It requests a powerful anti-inflammatory management with immunosuppressive drugs. Nevertheless, JIA children below 6 years of age or those with severe uveitis or a previous failure of implantation in the opposite eye should remain aphakic. Commercial Relationships: Damien Guindolet, None; Pascal Dureau, None; Catherine Edelson, None; Amandine Barjol, None; Céline Terrada, None; Georges Caputo, None; Phuc Lehoang, None; Bahram Bodaghi, None Program Number: 2509 Poster Board Number: C0029 Presentation Time: 3:45 PM–5:30 PM Clinical characteristics of autoimmune neuro-retinopathy (AINR) Eric Sollenberger1, Rebecca S. Epstein1, Grazyna Adamus2, Alessandro Iannaccone1. 1Ophthalmology/Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN; 2 Ocular Immunology Lab, Oregon Health & Science University, Portland, OR. Purpose: To illustrate the clinical and functional presentation of a retrospective case series of 57 patients with AINR. Methods: Age, onset modality, symptoms at onset, fundus features, Goldmann visual fields (GVF), flash electroretinogram (ERGs) and pattern-reversal visual evoked potentials (PVEPs) were reviewed. Anti-retinal and anti-optic nerve autoantibody (AAb) testing through western blots and immunohistochemistry (IHC) was performed in a diagnostic setting to confirm the suspicion of AINR. Results: Average patient age at first exam was 50±16.2 yo (SD) and as early as 4 yo. Patients were 63% female. Mean age of onset was 46.3±16.3 yo (SD). In most cases, onset was acute or subacute. Patients presented with as many as 6 anti-retinal and/or anti-optic nerve AAbs. Symptoms of night blindness (82%), decreased visual acuity (77%), photophobia (68%), and photopsia (49%) were common in AINR. On fundus exam, Papillary and juxtapapillary pigmentation and/or atrophy, disc elevation and/or hyperemia, temporal atrophy, and cupping were seen 93% of cases. Macular changes (epiretinal membrane, RPE changes, and focal macular atrophy) were common (85%). Peripheral punched-out retinal lesions (61%), bone spicule-like deposits (59%) and vascular attenuation, alongside vasculitic changes and sheathings (61%), were also frequently observed. PVEP delays were found in 87% of cases, also with 20/20 acuity. ERG abnormalities were found in 95% of the tested subjects. The most common findings were photopic ERGs ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts delays (86%) and reduced amplitude (75%). Inter-ocular asymmetry seen by PVEP (85%), ERG (77%) and GVF (62%) criteria was another characteristic of AINR. All but one patient had asymmetry on at least one of these tests, while 56% had asymmetry in both PVEP and ERG. Combining PVEP and ERG data, we found that 90% of the tested patients had simultaneous retinal and optic nerve involvement. Conclusions: AINR patients present with recognizable clinical and functional findings. Our findings indicate that the proportion of cases with simultaneous involvement of the retina and of the optic nerve, whether at the retinal ganglion cell (RGC)/ retinal nerve fiber layer (RNFL) and/or retrobulbar level, in patients with autoimmune retinopathy – hence, actually affected with AINR – is even higher than what we recently reported in a large, multi-center case series of patients (Adamus et al. J Ophthalmic Inflamm Infect 2011;1:111-21). Commercial Relationships: Eric Sollenberger, None; Rebecca S. Epstein, None; Grazyna Adamus, None; Alessandro Iannaccone, None Support: Research to Prevent Blindness, Inc. New York, NY (Physician Scientist Award to AI and unrestricted grant to UTHSC Ophthalmology/Hamilton Eye Institute) and NIDDK Short-Term Research Training Grant DK-7405-29 (ES) Program Number: 2510 Poster Board Number: C0030 Presentation Time: 3:45 PM–5:30 PM Autoimmune retinopathy in patients with birdshot retinochoroidopathy (BSRC) who are in remission Homaira A. Hossain, C Stephen Foster. Ophthalmology, Massachusetts Eye Research and Surgery Institution, Cambridge, MA. Purpose: To investigate the continued deterioration of vision in patients with BSRC who are in remission from active uveitis. Methods: We identified four patients who demonstrated continued deterioration in visual function despite long-term remission from active BSRC as evinced by absence of inflammation on clinical ophthalmoscopic examination and fluorescein angiogram. This visual decline resembled that of autoimmune retinopathy, with painless loss of vision and decline in visual acuity, abnormal electroretinography, and defects on visual field testing. Sera were tested for anti-retinal and anti-optic nerve antibodies by Western blot analysis. Results: The four patients included two males and two females, with an average age of 60.3 years (ranging from 54 to 68 years). All patients were HLA-A29 positive. Three patients were treated with a minimum of two years of immunomodulatory therapy (IMT). One patient was treated with IMT for five months, after which it was discontinued due to medication side effects. She subsequently underwent bilateral fluocinolone acetonide intravitreal implants. At the time of serum testing, all four patients demonstrated absence of inflammation on clinical ophthalmoscopic examination and fluorescein angiogram. Two patients demonstrated visual decline as evinced by both visual field testing (SITA-SWAP) and electroretinography (decreased amplitude and/or increased implicit time on 30 Hz flicker). One patient demonstrated visual decline in electroretinography testing only, and one patient demonstrated visual decline in visual field testing only. The serum of all four patients demonstrated positivity for anti-retinal antibodies. The serum of three out of four patients demonstrated positivity for anti-optic nerve antibodies. Conclusions: The presence of anti-retinal and anti-optic nerve antibodies in patients who have achieved long-term remission of BSRC after prolonged treatment suggests that continued deterioration has occurred because of autoimmune retinopathy. Patients with BSRC who present with visual decline but show no active inflammation on clinical ophthalmoscopic examination and fluorescein angiogram may benefit from autoantibody screening. For these patients, further treatment with IMT may be indicated due to the presence of autoantibodies implicated in the continued deterioration of visual function. Commercial Relationships: Homaira A. Hossain, None; C Stephen Foster, None Program Number: 2511 Poster Board Number: C0031 Presentation Time: 3:45 PM–5:30 PM VASCULAR ENDOTHELIAL GROWTH FACTOR IN TEARS OF SYSTEMIC SCLEROSIS PATIENTS Aniko Rentka. Department of Ophthalmology, University of Debrecen, Debrecen, Hungary. Purpose: Several cytokins, adhesion molecules and growth factors play key role in inflammatory processes of the eye. Quantitative changes of these factors are measurable of serum. In many cases changes in these patiens’ tears is presented. The essential role of vascular endothelial growth factor (VEGF) in the immunpathology of systemic sclerosis (SSc) is confirmed in blood serum previously. However, there have been no any other investigations according to VEGF level determination in tears of SSc patients. To determine VEGF and total protein levels in tears of SSc patients. We also aimed to define tear secretion velocity in these patients and correlated the levels with clinical signs and symptoms. Methods: Tear samples were collected from 43 SSc patients and 27 healthy controls using capillary micropipette, first without, then with stimulation. Tear collecting time was measured during tear secretion. Diluted samples were stored in polypropylene tubes at -80 °C until assessment. Total protein values were determined using the BCA Microplate method, and then VEGF levels were measured with R&D immunoassay kit. The protocol that was used in this study was in full compliance with good clinical practices, the Declaration of Helsinki (1996), and the guidelines of the Medical and Health Science Centre of the University of Debrecen. Results: Patients’ average tear secretion velocity was 4.53 ml/min (1.5-25.6). The average total protein value was 6.9 mg/ml (1.8-12.3) in case of basal tear secretion and 6.26 mg/ml (2.3-16.5) in tears collected after stimulation. In tears collected without stimulation the average VEGF level was 4.95 pg/ml (3.518.06) and 4.69 pg/ml (2.66-6.44) with stimulation. We measured 4.13 mg/ml (1.0-14.1) total protein and 6.15 pg/μl (3.84-12.3) VEGF level in tears of healthy controls. Conclusions: In patients with keratoconjunctivitis sicca, the most frequent ocular sign of SSc, tear secretion velocity was 67% of the controls’. Total protein values in patients were 42% higher than in healthy controls, it may mean that total protein production or since SSc patients have a decreased tear secretion velocity. The protein concentration is only increased because of the smaller tear volume. Though VEGF in tears of SSc patients is decreased with 20%, and did not change upon stimulation, it can be explained also with the decreased tear secretion of patients. Commercial Relationships: Aniko Rentka, None Program Number: 2512 Poster Board Number: C0032 Presentation Time: 3:45 PM–5:30 PM Intraocular rubella virus antibody detection as a diagnostic tool for atypical forms of Fuchs uveitis Aymeric Bouillot1, Audrey Fel1, Valerie Touitou1, Phuc Lehoang1, Flore Rozenberg2, Bahram Bodaghi1. 1Ophthalmology, DHU ViewMaintain, Pitie Salpetriere Hospital, Paris, France; 2Virology, Saint Vincent de Paul Hospital, Paris, France. Purpose: To analyze the clinical profile and prognosis of rubella virus-associated uveitis. ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Methods: All patients managed between January 2009 and August 2013 for an atypical form of Fuchs uveitis were included in this study. Clinical features for these patients were assessed. An extensive work-up of anterior or intermediate uveitis was performed in all cases. An anterior chamber tap was performed for molecular detection of herpesviruses and intraocular anti-rubella virus antibody synthesis. Treatment was adapted to the results of ocular fluids analysis. Results: The series included 27 patients (M/F : 14/13) with a proven diagnosis of rubella virus-associated uveitis. The mean age was 39.6 years (23-67). All patients had an intraocular antibody production against rubella virus with a Goldmann-Witmer coefficient > 3. Only 7 patients (23%) fulfilled the classical criteria of Fuchs cyclitis with stellar keratic precipitates, heterochromia/iris atrophy, cataract and vitritis. 15 patients (48%) had a posterior subcapsular cataract requiring surgery. Mean laser flare meter values were 13.8 ph/ms. 18 patients (58%) had vitritis. Uveitis was bilateral in 4 patients (15%) and 10 patients (32%) had an uncontrolled IOP requiring filtering surgery. Conclusions: For most of these patients, intraocular rubella virus antibody production allowed the final diagnosis of Fuchs uveitis despite incomplete clinical criteria. Intraocular rubella virus detection can help to establish the diagnosis of atypical forms of Fuchs cyclitis. This avoids unnecessary additional tests and prescription of local or systemic corticosteroids. Secondary glaucoma remains a major complication and needs an appropriate management in order to avoid a poor visual outcome. Commercial Relationships: Aymeric Bouillot, None; Audrey Fel, None; Valerie Touitou, None; Phuc Lehoang, None; Flore Rozenberg, None; Bahram Bodaghi, None Program Number: 2513 Poster Board Number: C0033 Presentation Time: 3:45 PM–5:30 PM Scleritis in Australia: Disease Associations and Ocular Complications Julie L. Morrison1, Ethan Nguyen3, 4, Jessica Brennan1, Richard Stawell2, Lyndell L. Lim1, 2, Peter J. McCluskey4. 1Centre for Eye Research Australia, University of Melbourne, East Melbourne, VIC, Australia; 2Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; 3Sydney Eye Hospital, Sydney, NSW, Australia; 4 Save Sight Institute, Sydney Medical School University of Sydney, Sydney, NSW, Australia. Purpose: Scleritis is a rare ocular inflammatory condition which has the potential for significant visual loss. This retrospective review looks at the demographics, clinical features and complications associated with infectious and non-infectious scleritis in an Australian population. Methods: Retrospective chart review of 90 patients with scleritis from three Australian tertiary referral centers from 1988 to 2013. Data collected included type of scleritis, demographics, ocular complications, associated systemic disease and treatments. Results: The mean age of onset was 50 years of age (range 8 to 85). Females accounted for 62% of presentations and had an earlier mean age of onset (46 vs. 57, p=0.01).Nine of the 90 subjects had infectious scleritis (tuberculosis n=3, herpes zoster ophthalmicus n=2, other n=4). Of the non-infectious scleritis, the commonest form was diffuse (45%), followed by nodular (23%), necrotising (16%) and posterior (16%). The majority of cases (75%) were unilateral, with diffuse scleritis being more likely to be bilateral than other types (39%,p=0.008). Forty percent were found to have an associated systemic disease, with diffuse anterior scleritis the most likely to be associated with systemic disease than any other type of scleritis (45%). The most commonly associated systemic disease was rheumatoid arthritis (RA, n=9, 29%), followed by Wegener’s granulomatosis (n=5, 16%). Those with RA were more likely to have bilateral involvement (56%, p=0.037). Necrotising disease was more likely than non-necrotising disease to have associated corneal involvement (p=0.019), cataract (p=0019) and keratitis (p=0.032). Those with posterior involvement were more likely to have reduced visual acuity (p=0.010), due to its higher association with uveal effusion (p=0.012), macular edema (p=0.028) and retinal detachment (p<0.001). Conclusions: This is the first study to categorize an Australian population of scleritis patients. Consistent with previous studies, we found females were more likely to suffer from scleritis and present at a younger age. Necrotizing scleritis or scleritis with posterior involvement were more likely to be associated with ocular complications. A higher proportion of posterior scleritis was identified compared to previous studies. Commercial Relationships: Julie L. Morrison, None; Ethan Nguyen, None; Jessica Brennan, None; Richard Stawell, None; Lyndell L. Lim, None; Peter J. McCluskey, None Program Number: 2514 Poster Board Number: C0034 Presentation Time: 3:45 PM–5:30 PM Assessment of Inflammatory Cytokines in Aqueous Humour of Patients with Sarcoidosis Kyungmin Lee1, 2, Abhishek R. Payal1, 3, Jamie Metzinger1, Reena Rasheed1, Ninani Kombo1, Pranav Patel1, Homaira A. Hossain1, C Stephen Foster1, 4. 1Massachusetts Eye Research and Surgery Institution, Cambridge, MA; 2Retina Center, HanGil Eye Hospital, Incheon, Republic of Korea; 3Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA; 4Harvard Medical School, Boston, MA. Purpose: To compare the concentrations of 20 cytokines from T helper (Th) pathways (Th1, Th2, and Th17) in aqueous humour of patients with sarcoid uveitis (active or in remission) to normal eyes. Methods: Single center, prospective, observational case control study. Aqueous samples were collected from 7 eyes with sarcoid uveitis (SU group), and 8 normal eyes (control group). Quantitative multiplex sandwich ELISA-based microarray assay was used to quantify 20 cytokines: interleukin (IL)-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, IL-17f, IL-21, IL-22, IL-23, IL-28a, macrophage inflammatory protein-3α (MIP-3α), transforming growth factor-β1 (TGF-β), interferon-γ (IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α) and tumor necrosis factor-β (TNF-β). Cytokine levels from patients were compared with normal controls. Results: The aqueous levels of IL-2 (10.6 pg/ml: 1.5-40.5, p: 0.024) and TNF-α (98.5 pg/ml: 4.2-542.6, p: 0.0093) were significantly higher in the sarcoidosis group compared with the control group. The aqueous levels of IL-2, IL-6, IL-21, IL-23, and GM-CSF were higher in the active uveitis patients compared to the inactive group. TNF-α was higher in the remission patients. Conclusions: Multiplex immunoassay of 20 cytokines in patients with sarcoid associated uveitis revealed that IL-2 and TNF- α were concentrated in the aqueous fluid. We confirm that IL-21 and IL-23 levels were elevated and correlated with disease activity, suggesting Th17 cells play an important role in autoimmune disease and uveitis. Commercial Relationships: Kyungmin Lee, None; Abhishek R. Payal, None; Jamie Metzinger, None; Reena Rasheed, None; Ninani Kombo, None; Pranav Patel, None; Homaira A. Hossain, None; C Stephen Foster, None ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Program Number: 2515 Poster Board Number: C0035 Presentation Time: 3:45 PM–5:30 PM Long-Term Drug-Free Remission and Visual Outcomes in Sympathetic Ophthalmia Abhishek R. Payal1, 2, C Stephen Foster2, 3. 1Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA; 2Massachusetts Eye Research and Surgery Institution, Cambridge, MA; 3Harvard Medical School, Boston, MA. Purpose: Sympathetic ophthalmia (SO) has an unpredictable but often chronic course requiring prolonged corticosteroid or immunosuppressive therapy (IST) or a combination of both. The therapy itself may result in additional complications in the remaining good eye. We assessed corticosteroid and IST free long-term remission in the treatment of patients with sympathetic ophthalmia. Methods: Retrospective case series of 19 patients with sympathetic ophthalmia treated with corticosteroids and immunosuppressive therapy with a step-ladder protocol practiced at this center between March 2005-October 2013. Primary outcomes were five-year and two-year remission rates with and without corticosteroids and immunosuppressive therapy; and visual acuity. Results: Nineteen patients (10 females, 9 males) with sympathetic ophthalmia, meeting inclusion criteria were identified for review. Patients were in the age range of 16.1-94.95 years (median age 58.56 years). Median follow-up was 7.10 years (mean, 6.41; range, 2.5 to 8.63 years). All patients achieved remission, thirteen of them for 2 years or more. Three (15.78%) of the 19 patients maintained remission off all immunosuppressive therapy and corticosteroids for more than 5 years with vision of 20/25 or better in the sympathizing eye. Thirteen patients (68.42%) were in remission on immunosuppressive therapy or corticosteroids or in combination therapy. Eleven patients (57.9%) maintained visual acuity of 20/40 or better at the end of follow up. Conclusions: Even with a devastating and possibly lifelong disease like sympathetic ophthalmia, long-term remission off all immunosuppressive therapy and corticosteroids, perhaps even cure, is possible. It is worthwhile to consider immunosuppressive therapy early in the management of sympathetic ophthalmia for better outcomes. Studies with longer follow-up might provide additional evidence to support this. Commercial Relationships: Abhishek R. Payal, None; C Stephen Foster, None Program Number: 2516 Poster Board Number: C0036 Presentation Time: 3:45 PM–5:30 PM Duration of Immunomodulator Therapy and Uveitic Relapse David Mostafavi, Michael Chang, Vicente Diaz, John Mauro, Sanjay Kedhar, Michael Samson. New York Eye and Ear Infirmary, New York, NY. Purpose: To evaluate the relationship of length of time on immunomodulator therapy (IMT) and relapse of uveitis upon IMT discontinuation. Methods: 1100 charts were reviewed at New York Eye and Ear Infirmary from the years 2004-2006. Inclusion criteria included patients who were on Methotrexate or Cellcept for at least 1 year and had a minimum 3 year follow-up period once the medication was discontinued. Three groups were formed: those who were on IMT for 1-2 years, 2-3 years, and greater than 3 years. The first 30 patients who met the inclusion criterion were included in each group. The length of time from IMT discontinuation to relapse was noted. Results: About half of the patients in the 1-2 year IMT group eventually had a relapse. There was no statistically difference between the 2-3 year and 3 year or greater IMT group. Conclusions: This pilot study suggests that patients should be treated for a minimum of two years with IMT to minimize the risk of uveitic relapse in the future. Commercial Relationships: David Mostafavi, None; Michael Chang, None; Vicente Diaz, None; John Mauro, None; Sanjay Kedhar, None; Michael Samson, None Program Number: 2517 Poster Board Number: C0037 Presentation Time: 3:45 PM–5:30 PM Autoimmune Retinopathy Treated with the Fluocinolone Acetonide Intravitreal Implant After Intolerance to Systemic Immunosuppression David A. DiLoreto, Zoë Williams, Yousuf M. Khalifa. Ophthalmology, Flaum Eye Institute, U Rochester Med Ctr, Rochester, NY. Purpose: To report outcomes of two cases of autoimmune retinopathy (AIR), one cancer associated retinopathy (CAR) and one non-paraneoplastic autoimmune retinopathy (npAIR), treated locally with the fluocinolone acetonide intravitreal implant (Retisert) due to intolerance to systemic immunosuppression Methods: Case reports. Results: Case 1. A 64 year old male with CAR secondary to poorly differentiated carcinoma of the lung (confirmed with markedly diminished full-field ERG and anti-retinal autoantibody testing positive for antibodies against 30-kDA (carbonic anhydrase II), 36-kDa (GAPDH), 40-kDa (aldolase), 45-kDa (arrestin) and 62-kDa proteins) was treated with a sustained release intravitreal steroid implant (fluocinolone acetonide 0.59mg (Retisert)) after progressive vision loss despite intravenous immunoglobulin therapy. He was unable to tolerate systemic immunosuppression with mycophenolate mofetil due to leucopenia and developed multiple adverse side effects from high dose prednisone. His vision was successfully stabilized with a sustained release intravitreal fluocinolone acetonide implant alone for 6 months prior to his death. Case 2. An 87 year old male with a 10-year history of chronic bilateral uveitis, severe arteriolar attenuation and bilateral visual field constriction was diagnosed with npAIR (antiretinal antibody testing pending) after work-up failed to reveal cancer. Optical coherence tomography scan revealed diffuse thinning of the retina with limited sparing of the inner segment/ outer segment junction in the central macula. He had been treated previously with topical and subtenon’s steroids with limited response. Intravitreal steroid injections stablilized the disease for limited duration. Treatment with mycophenolate mofetil was not tolerated and he deferred trial of other immunosuppressive agents. Bilateral intravitreal fluocinolone acetonide implants were used to stabilize his disease at 20/150 in both eyes with more than one year of follow-up. Conclusions: Local ocular treatment with steroids is an option in patients with autoimmune retinopathy when systemic immunosuppresion fails or is not tolerated. We report the first use of intravitreal steroids in sustained release form to treat autoimmune retinopathy without concomitant use of systemic immunosuppression. Commercial Relationships: David A. DiLoreto, None; Zoë Williams, None; Yousuf M. Khalifa, Alcon (R), Bausch & Lomb (R) Support: Research to Prevent Blindness ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Program Number: 2518 Poster Board Number: C0038 Presentation Time: 3:45 PM–5:30 PM Uveitis Reactivation in Children Treated with Tumor Necrosis Factor-α Inhibitors Michael Lewen3, Monte Mills1, Melissa Lerman2. 1Division of Ophthalmology, The Children’s Hospital of Philadelphia, Philadelphia, PA; 2Division of Rheumatology, The Children’s Hospital of Philadelphia, Philadelphia, PA; 3University of Pennsylvania Health System, Philadelphia, PA. Purpose: To evaluate reactivation of pediatric uveitis following treatment with TNF-alpha inhibition (anti-TNFα). Methods: We retrospectively assessed children (≤18 years) with non-infectious uveitis who achieved quiescence while receiving anti-TNFα at The Children’s Hospital of Philadelphia (January, 2000-July, 2012). Reactivation was defined as redevelopment of “active” uveitis. Kaplan-Meier (KM) methods were used to calculate the rates of reactivation while still on treatment (primary outcome) and following complete discontinuation of anti-TNFα (secondary outcome). Variables were evaluated for their association with failure (reactivation). Results: Of the 136 children and adolescents treated for uveitis, 39 met criteria for assessment of the primary, and 19 met criteria for assessment of the secondary outcome. Almost two thirds of subjects were female, almost half carried systemic diagnoses of Juvenile Idiopathic Arthritis, and the majority were treated with infliximab. The KM estimated proportion who failed within 12 months was 27.8% (95% confidence interval [95% CI]: 15.9-45.8%). The estimated probability of failure within 12 months was higher following discontinuation of anti-TNFα (63.8% [95% CI: 38.987.7%]) relative to before discontinuation (21.6% [95% CI: 10.840.2%]). Amongst those who discontinued anti-TNFα, the likelihood of failure was significantly lower for those treated with infliximab (vs. adalimumab) (Hazard Ratio [HR] 0.07, 95% CI: 0.01-0.46). Older age at onset of ocular disease was associated with a significant increase in the hazard of failure (HR 1.32, 95% CI: 1.03-1.69). The duration of remission-on-medication did not significantly affect the likelihood of failure. Conclusions: The durability of the response to anti-TNFα is limited in those children who remain on drug following achievement of uveitis quiescence. It is poorly sustained in those who discontinue anti-TNFα - with the majority having uveitis reactivation. These data suggest that infliximab results in a more durable remission-offmedication than does adalimumab. Results from this small cohort did not support the notion that maintenance of remission-on-medication for a longer duration decreases the likelihood of later reactivation. Commercial Relationships: Michael Lewen, None; Monte Mills, None; Melissa Lerman, None Program Number: 2519 Poster Board Number: C0039 Presentation Time: 3:45 PM–5:30 PM : Use of Intravenous pulses of cyclophosphamide treatment in the control of severe ocular inflammatory disease Roberto Dalli1, Miguel Pedroza-Seres1, 2, Vanessa Valderrama2. 1 Uveitis and Ocular Immunology, Retina Clinic, Guadalajara, Mexico; 2Uveitis and Ocular Immunology, Institute Of Ophthalmology Conde de Valenciana, Mexico city, Mexico. Purpose: The goal of this Study was the evaluation of Intravenous pulses of cyclophosphamide in the control of severe ocular inflammatory disease. Methods: A total of 20 patients were included in this study. All the patients received Intravenous pulses of cyclophosphamide (10-15 mg/ kg). Review of systems and a complete ophthalmologic exam were made in each patient. The severity of their disease was considered in order to treat them with intravenous cyclophosphamide. Results: From a total of 20 patients, 14 (70%) were female and 6 (30%) were male. The average age of patients was 51.35 ± 22.22 years old. Ocular findings were necrotizing scleritis (30%), followed by nodular scleritis (10%), difuse scleritis (10%) and others as retinal vasculitis, mucous membrane pemphigoid, rheumatoid arthritis. The average number of pulses of cyclophosphamide were 3.5 ± 2.5. All patients were treated with oral steroids. Ninety percent of patients showed improvement in the course of the disease. Seventy per cent of patients had systemic disease, granulomatosis with polyangeitis, (Wegener’s disease) was seen in 35%, ocular cicatrizial pemphigoid (10%), tubercoulosis disease (10%), rheumatoid arthritis (5%) and miscellaneous (5, 5%). The ocular manifestation in patients was primarily the scleritis. Conclusions: Use of intravenous pulses of cyclophosphamide in acute ocular inflammatory disease is effective and safe. It should be considerer in any patient with a potential blinding inflammatory disease Commercial Relationships: Roberto Dalli, None; Miguel PedrozaSeres, None; Vanessa Valderrama, None ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2014 Annual Meeting Abstracts Program Number: 2520 Poster Board Number: C0040 Presentation Time: 3:45 PM–5:30 PM Combination of Rituximab and Intravenous Immunoglobulin for Recalcitrant Ocular Cicatricial Pemphigoid Asima Bajwa, Pranav Patel, Judit Z. Baffi, Ninani C. Kombo, Homaira A. Hossain, Kyungmin Lee, C Stephen Foster. Massachusetts Eye Research and Surgery Institution, Cambridge, MA. Purpose: To analyze the clinical course of patients with recalcitrant Ocular Cicatricial Pemphigoid (OCP) treated with Rituximab (RTX) and intravenous immunoglobulin (IV-Ig) Methods: A non comparative retrospective case series of 12 immunohistopathologically proven OCP patients who failed conventional immunosuppressive therapy. All patients received a combination of RTX and IV-Ig according to the protocol and a minimum follow up of 6 months. The main outcome measures were blindness (best corrected visual acuity BCVA 20/200 or worse), control of inflammation and Foster OCP staging. RTX + IV-Ig Treatment Protocols: 1) RTX: Dose 375mg/m2 once weekly for 8 consecutive weeks, then monthly for the subsequent 4 months, total of 12 infusions during a period of 6 months. 2) IV-Ig: Dose 2g/kg/cycle. Total dose was divided into 3 equal parts on 3 consecutive days. The patients were given one cycle of IV-Ig prior to the initiation of RTX and then monthly until the B cell count returned to normal. Thereafter, IV-Ig was continued at 6, 8, 10, 12, 14 and 16 weeks. The last cycle was given at 16 week interval. Results: Twelve OCP patients (24 eyes), 7 female and 5 male, with a median age of 58 years (range 40–70 years) were included. The duration of the disease varied from 2-168 months (median 24). Total follow-up period was between 6-108 months (median 18 months. All but 5 patients received the above RTX+IV-Ig protocol (median 12.5 infusions). Five patients with aggressive disease (2 were monocular, and 1 relapsed after being in remission for a year) received additional RTX infusions. Of these 1 eventually failed therapy. The duration of RTX infusions was 4-19 months (median 12months). None of the eyes became blind at the end of follow up. Eight patients (66.6%) were in remission after 3-12 months (median 6months). Of these 8 patients, 6 achieved remission off therapy while 2 (16.6%) had resolving inflammation with continuation of therapy. Two patients failed (16.6%). Before and after therapy, OCP staging remained stable throughout the study period. Conclusions: Combination of RTX and IV-Ig is effective in preventing blindness and arresting disease progression in patients with recalcitrant OCP. Commercial Relationships: Asima Bajwa, None; Pranav Patel, None; Judit Z. Baffi, None; Ninani C. Kombo, None; Homaira A. Hossain, None; Kyungmin Lee, None; C Stephen Foster, None ©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].