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ARVO 2012 Annual Meeting Abstracts 530 Autoimmune Ocular Disease Thursday, May 10, 2012, 8:30 AM - 10:15 AM Hall B/C Poster Session Program #/Board # Range: 6228-6256/D1088-D1116 Organizing Section: Immunology/Microbiology Contributing Section(s): Cornea,Retina+,Retinal Cell Biology Program Number: 6228 Poster Board Number: D1088 Presentation Time: 8:30 AM - 10:15 AM Intraocular Th1/Th17 Cells Coexpressing IL-10: Tregs that Prevent Recurrent EAU? Ulrike Kaufmann, Maria Diedrichs-Moehring, Gerhild Wildner. Section of Immunobiology, Ophthalmology, Clinic of the Ludwig-Maximilians-University, Munich, Germany. Purpose: The strict compartmentation of T cells in Th1, Th2 and Th17 was mitigated with the discovery of T cells that simultaneously produce IFN-γ and IL17. They are found in chronically inflamed tissues and are not stable with respect to their cytokine expression. The role of these cells is still unknown. We have investigated intraocular T cells during the course of EAU with respect to their cytokine and Foxp3 expression in our rat model of monophasic and relapsingremitting uveitis. Methods: After induction of EAU with PDSAg (monophasic EAU) or R14 (relapsing EAU) in CFA we collected intraocular cells at various time points during ocular inflammation and stained them ex vivo for TCRαβ and intracellular IFN-γ, IL-17, IL-10 and Foxp3 expression. Results: During the course of PDSAg-induced, monophasic uveitis intraocular T cell populations coexpressing IFN-γ and IL-17 increased from onset via peak to remission of EAU. IL-10+ and IL-17+/IL-10+ cells displayed a similar population dynamic, the latter increased to about 30% of the Th17 population at remission. Like Foxp3+ cells, IFN-γ+/IL-10+ cells were a minor population remaining stable during the course of monophasic EAU. In R14-induced relapsing EAU the number of IL-17+ and IFN-γ+/IL-17+ cells decreased at remission, while IFN-γ+ cell numbers remained elevated. IL-17+/IL-10+ cells decreased during remission of R14-induced EAU, while IFN-γ+/IL-10+ and Foxp3+ cells were also minor intraocular lymphocyte populations. Conclusions: The change of the intraocular T cell populations during EAU, the large numbers of T cells producing multiple cytokines and the differences observed between monophasic and relapsing disease points to a strong population dynamics in the eye, potentially influencing the course of EAU. The strong increase of IL10+ T cells in the eyes during monophasic EAU suggests a regulatory role of these cells with a potential to prevent relapses. Foxp3+ cells seem to play no role in preventing recurrent EAU. Commercial Relationships: Ulrike Kaufmann, None; Maria DiedrichsMoehring, None; Gerhild Wildner, None Support: Deutsche Forschungsgemeinschaft SFB 571 Program Number: 6229 Poster Board Number: D1089 Presentation Time: 8:30 AM - 10:15 AM Cd4+ Foxp3+ Cd25Bright T Regulatory Cells Population In Ocular Sarcoidosis Alexis Pinel1A, Alexis Mathian1B,2, Makoto Miyara1B, Catherine CHAPELONABRIC1B, Christophe Parizot1C, Du Boutin1B, Zahir Amoura1B, Guy Gorochov1C, Phuc Lehoang1A, Bahram Bodaghi1A,3. AOphthalmology, BInternal medecine, C Immunology, 1CHU Pitie-Salpetriere, Paris, France; 2INSERM UMR-S 945, Paris, France; 3U972 INSERM, Paris, France. Purpose: CD4+ FOXP3 CD25bright T regulatory cells (Treg) are expanded in systemic sarcoidosis wich is a chronic inflammatory disorder characterized by noncaseating granulomas. The effector Treg expansion is already described in pulmonary and renal sarcoidosis. The main objective of this study is to describe the circulating Treg population (naive and effector Treg) in uveitis and in particular in ocular sarcoidosis. Methods: Patients followed for uveitis were included. Clinical, biological and pathological features were analyzed. The patients were divided in 4 groups : definite ocular sarcoidosis among the international criteria for the diagnosis of ocular sarcoidosis from the International Workshop on Ocular Sarcoidosis (IWOS), suspected ocular sarcoidosis but not biopsy proved, idiopathic uveitis and uveitis of other origin. Treg populations were measured in blood using fluocytometry analysis. Results: 103 patients were included with 19 definite ocular sarcoidosis, 12 suspected sarcoidosis, 41 idiopathic uveitis and 31 uveitis of other diagnosis. In the definite ocular sarcoidosis and the suspected sarcoidoisis groups, the median effector Treg rate was 2,54 and 2,7 which was significantly higher than in the control group (p<0,0001). There were no statistical difference between the Treg effector rate of idiopathic uveitis (0,86), uveitis of other diagnosis (0,73) and the control group. In the definite ocular sarcoidosis group, the Treg was significantly higher compared to idiopathic uveitis (p=0,046) and to uveitis of other origin (p=0,009). The naive Treg population showed no statistical difference between the 4 groups. Conclusions: Definite ocular sarcoidosis and suspected ocular sarcoidosis are associated with peripheral effector Treg expansion. There is no alteration of Treg population in idiopathic uveitis and uveitis of other origin. The naive Treg population is not expanded in the 4 groups. This test could be used as a new diagnostic tool in uveitis in particular when sarcoidosis is suspected. Commercial Relationships: Alexis Pinel, None; Alexis Mathian, None; Makoto Miyara, None; Catherine Chapelon-abric, None; Christophe Parizot, None; Du Boutin, None; Zahir Amoura, None; Guy Gorochov, None; Phuc Lehoang, None; Bahram Bodaghi, None Support: None Program Number: 6230 Poster Board Number: D1090 Presentation Time: 8:30 AM - 10:15 AM Assessment of Th1, Th2, and Th17 Cells in Birdshot Retinochoroidopathy Paul Yang, C. S. Foster. Ophthalmology, Massachusettes Eye Research and Surgery Institute, Cambridge, MA. Purpose: To correlate the serum levels of 20 cytokines from the T helper (Th)1, Th2, and Th17 pathways with disease activity in birdshot retinochoroidopathy (BSRC). Methods: Single center cohort study of 4 BSRC patients, and 4 normal controls. Immunomodulatory therapy (IMT) and disease status were recorded for each patient. Disease activity was assessed with visual acuity, visual field, fundus exam, optical coherence tomography, fluorescein angiography, and electroretinography. Sera were collected, and a quantitative multiplex sandwich ELISA-based microarray assay was used to quantify 20 cytokines: interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, IL-17F, IL-21, IL-22, IL-23, IL28, interferon (IFN)-gamma, macrophage inflammatory protein (MIP)-3alpha, transforming growth factor (TGF)-beta1, tumor necrosis factor (TNF)-alpha, TNFbeta, and granulocyte macrophage colony-stimulating factor (GM-CSF). Cytokine levels from patients were compared with normal controls and correlated with IMT and disease activity. Results: IL-21 (p = 0.01), IL-23 (p = 0.02), TGF-beta1 (p = 0.02), and TNF-beta (p = 0.02) were significantly elevated in the sera of BSRC patients compared to normal controls. IL-17 (p = 0.31) and IFN-gamma (p = 0.85) serum levels were not significantly elevated. The subject that had been in IMT-dependent clinical remission for 2 years, had the lowest levels of IL-21 and IL-23 levels. IL-21 and IL-23 levels had a correlation coefficient of r = 0.93. Conclusions: Th17 cells play an important role in autoimmune disease and uveitis. Although standard ELISA assay has not shown elevations of IL-17 in the sera of BSRC patients, a recent multiplex immunoassay of 23 cytokines in BSRC patients revealed that IL-17 and proinflammatory cytokines were concentrated in the aqueous fluid. We confirm that IL-17 is not elevated in the sera of BSRC patients, however both IL-21 and IL-23 levels were significantly elevated and correlated, suggesting that peripheral cytokines may promote Th17 cells in an organ specific manner. In addition, higher IL-21 and IL-23 levels may correlate with active disease. Commercial Relationships: Paul Yang, None; C. S. Foster, None Support: Ocular Immunology and Uveitis Foundation Program Number: 6231 Poster Board Number: D1091 Presentation Time: 8:30 AM - 10:15 AM Regulatory T-Cells In Peripheral Blood of Patients With Birdshot Retinochoroidopathy Sana S. Siddique1, Laura Amorese1, Lama Almulki2, Ana M. Suelves3, C Stephen Foster4. 1Ophthalmology, MERSI/OIUF, Cambridge, MA; 2Ophthalmology, Massachusetts Eye Res and Surgery Inst, Cambridge, MA; 3MERSI, Xirivella, Valencia, Spain; 4Ophthalmology, Ocular Immunol & Uveitis Fndtn, Cambridge, MA. Purpose: CD4+CD25 T cells have been shown to be involved in the pathogenesis of autoimmune diseases. We aimed to assess the percentage of T regulatory cells in the peripheral blood of patients with active Birdshot Retinochoroidopathy (BSRC). Methods: After obtaining informed consent, 5ml of blood was collected from a total of twelve patients diagnosed with new onset or untreated BSRC and 5 healthy controls. The samples were labeled and transported immediately to the Clinical Flow Cytometry Laboratory at the Massachusetts General Hospital (MGH) for analysis. Peripheral blood mononuclear cells were subjected to flow cytometry for analysis of lymphocytes expressing the following cell surface glycoproteins: CD4 CD25 CD127 FOXP3. The biomarkers utilized to appropriately identify and purify activated T-regulatory cells included CD4, CD25, CD127, Foxp3, CD3 and CD45. Results: Samples were analyzed from 12 active BSRC patients and 5 healthy controls. The preliminary data reveals level of activated T-cells CD4+CD25hi was 4.6% in patients and 5.1% in the controls, while the level of CD4+CD25hi FOXP3+ regulatory T lymphocytes levels was 80.2% in patients and 78.4% in controls. Conclusions: Our preliminary data shows no statistical difference between the two Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts groups. It is unclear whether CD4+CD25hi FOXP3+ Treg cells play a role in the development of BSRC. Commercial Relationships: Sana S. Siddique, None; Laura Amorese, None; Lama Almulki, None; Ana M. Suelves, None; C Stephen Foster, None Support: None Program Number: 6232 Poster Board Number: D1092 Presentation Time: 8:30 AM - 10:15 AM Clinical Course of Patients with Behcet's Uveoretinitis that Discontinued Infliximab Therapy Tatsushi Kawaguchi1,2, Yuko Iwasaki1,2, Sayaka Kanda2, Sunao Sugita2, Manabu Mochizuki2. 1Ophthalmology, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan; 2Ophthalmology & Visual Science, Tokyo Medical and Dental University, Tokyo, Japan. Purpose: To investigate clinical course of patients with Behçet's uveoretinitis that discontinued infliximab (IFX) therapy. Methods: Subjects were uveitis patients associated with Behçet’s disease that received IFX treatment at Tokyo Medical and Dental University Hospital between 2000 and 2011. Medical records of patients who discontinued IFX were reviewed retrospectively. The patients followed less than 12 months before and after IFX treatment were excluded. Frequency and severity of acute uveitis attacks, and visual prognosis were evaluated. Results: Of forty patients included in this study, nine patients (22.5%) discontinued IFX due to adverse events. In these patients, frequency of acute uveitis attack (mean/month) was 0.56 before IFX, 0.22 during IFX, and 0.06 after cessation of IFX. The frequency of uveitis attacks was statistically higher before IFX compared to during IFX (p<0.01), and after cessation of IFX (p<0.01), while there was no statistical significance between during IFX and after cessation of IFX (p=0.12). The severity of uveitis attacks was tend to be higher in the periods before IFX compared to during IFX, or after cessation of IFX. The average of visual acuity was 0.25 before IFX, 0.30 during IFX, 0.37 at 12 months after cessation of IFX, and there was no statistical significance between them. Conclusions: Clinical course of Behçet's uveoretinitis after cessation of IFX appears to be satisfactory with well-controlled ocular inflammation. These data suggest a possibility for secure, planned discontinuation of IFX therapy in Behçet's uveoretinitis. Commercial Relationships: Tatsushi Kawaguchi, None; Yuko Iwasaki, None; Sayaka Kanda, None; Sunao Sugita, None; Manabu Mochizuki, None Support: None Program Number: 6233 Poster Board Number: D1093 Presentation Time: 8:30 AM - 10:15 AM Anti-DEC205 Mediated Delivery of Self-Antigen to Dendritic Cell Restores Tolerance in Spontaneous EAU Koju Kamoi1, Cristina Martin-Granados1, Corina Bobu1, Matthew E. Wikstrom2, Mariapia A. Degli-Esposti2, Ralph M. Steinman3, John V. Forrester1. 1 Ophthalmology, University of Aberdeen, Aberdeen, United Kingdom; 2Lion Eye Institute, University of Western Australia, Nedlands, Western Australia, Australia; 3 Rockfeller University, New York, NY. Purpose: Dendritic cells (DCs) play critical roles in the adaptive immune response participating both in immune activation and induction of tolerance. As an immunotherapy, DC “vaccination” has been used in patients with cancer for induction of immunity, but to date induction of tolerance for autoimmune disease is still at a preclinical stage. We have previously induced tolerance to IRBP by using antigen-pulsed immature DC and LPS-stimulated DC in a model of experimental autoimmune uveoretintitis (EAU). Here we have used a more targeted approach to induce tolerance by delivering antigenic peptide to different DC subsets using DCselective antibody-peptide fusion proteins in a spontaneous model of EAU. Methods: 1.Spontaneous EAU mice was induced in B10.BR mice by crossing single Tg 3A9 TCR mice with Tg mice expressing hen egg lysozyme (HEL) in photoreceptor cells under control of the promoter for IRBP. 2.Anti-DEC205-HEL fusion protein and anti-33D1-HEL fusion proteins were purified by the transfection of expression vectors. 3. Targeting of the fusion proteins to DC after subcutaneous injection was confirmed by immunostaining and flow cytometry of draining lymph node cells. 4. Modulation of spontaneous EAU was evaluated clinically and histologically. 5. Induction/expansion of regulatory T cells in eye and lymph node after fusion protein treatment were measured by flow cytometry. Results: Anti-DEC205-HEL and anti-33D1 fusion proteins (5ug subcut) tracked selectively to DLN DC. Administration of anti-DEC205-HEL at d18 postnatal prevented development of EAU, which reached maximum levels at around d 30 in 100% of untreated mice. In contrast, anti33D1-HEL led to an acceleration of EAU development. Histological evaluation indicated that anti-DEC205-HEL markedly reduced the numbers of infiltrating effector CD4+ T cells in the eye, while CD4+CD25+FoxP3+ regulatory T cells in eye-draining lymph nodes were increased by anti-DEC205-HEL, but decreased by anti-33D1-HEL. Conclusions: Targeted delivery of antigen to specific DC subsets can promote tolerance or immunity depending on the DC specific surface molecule. Clinical trails in promoting immune response to tumour antigens are already in progress. The data presented here in a preclinical model reveal the potential for such therapy to regulate autoimmune disease. Commercial Relationships: Koju Kamoi, None; Cristina Martin-Granados, None; Corina Bobu, None; Matthew E. Wikstrom, None; Mariapia A. DegliEsposti, None; Ralph M. Steinman, None; John V. Forrester, None Support: None Program Number: 6234 Poster Board Number: D1094 Presentation Time: 8:30 AM - 10:15 AM Monocyte-derived Macrophages in EAU Resolution Inbal Benhar1, Anat London1, Rachel R. Caspi2, Michal Schwartz1. 1Neurobiology, Weizmann Institute of Science, Rehovot, Israel; 2Laboratory of Immunology, National Eye Inst/NIH, Bethesda, MD. Purpose: We recently showed that monocyte-derived macrophages take part in protecting retinal ganglion cells (RGCs) after glutamate-induced retinal insult, by locally exerting an immunoregulatory phenotype. In experimental autoimmune uveitis (EAU), macrophages are commonly associated with disease induction and progression. Appreciating the functional diversity of monocyte-derived macrophages, we set out to find whether the same subsets of monocyte-derived macrophages that are involved in homeostasis restoration following glutamate intoxication are also involved in protection against immune-mediated damage associated with EAU. Methods: EAU was induced in male C57BL/6J mice by injection of human interphotoreceptor retinoid binding protein (IRBP)-derived peptide 1-20. The infiltration of monocyte-derived macrophages to the eye along disease course was monitored in CX3CR1-GFP bone marrow chimeric mice. Depletion experiments were performed in order to evaluate EAU resolution in the absence of monocytederived macrophages. Results: EAU induction resulted in the appearance of a distinct myeloid population in the retina. Monocyte-derived macrophages infiltrated diseased retinas, and were absent from retinas of control mice. Inhibition of this infiltration at the resolution phase resulted in a decrease in FoxP3+ regulatory T cells, and affected the local cytokine milieu. Conclusions: Monocyte-derived macrophages are active cells in EAU. These cells are present throughout disease course, affecting the local milieu in terms of cytokines and accumulation of other immune cells, and may play a role in disease resolution that is apparently distinct from their involvement in disease induction. Commercial Relationships: Inbal Benhar, None; Anat London, None; Rachel R. Caspi, None; Michal Schwartz, None Support: None Program Number: 6235 Poster Board Number: D1095 Presentation Time: 8:30 AM - 10:15 AM Alpha-1 Adrenergic Stimulation Exacerbates Acute Ocular Inflammation Through A Mechanism Mediated By Transforming Growth Factor Beta (TGFB) Paola A. Durand1, Yaohong Tan2, Darakshan Fatmi1, Xiaomei Xia2, Egla Suarez1, Victor L. Perez2, Jose L. Vega1. 1Neurology, Herbert Wertheim College of Medicine-Florida International University, Miami, FL; 2Ophthalmology, Bascom Palmer Eye Insitute, Miller School of Medicine, Miami, FL. Purpose: Previously we demonstrated that ocular immune privilege is abolished by the surgical transection of ocular sympathetic nerves. Here we investigated the effect of sympathetic adrenergic neurotransmission on acute ocular inflammation. Methods: The selective alpha-1 adrenergic receptor (A1AR) agonist PHE (5%) was applied every 8h for 24h to the ocular surface of mice afflicted with endotoxininduced uveitis. In some experiments topical prazosin (PRAZ), an A1AR blocker, was applied concomitantly in order to test the specficity of A1AR-mediated effects. Inflammation was quantified by flow cytometry of digested ocular homogenates using antibodies against cell surface markers known to appear in the context of acute inflammatory responses. Total aqueous humor protein quantification (via Bradford assay), and H&E staining were also performed to further characterize inflammation in EIU. In addition, we tested the effect of A1AR stimulation on neutrophilic infiltration following syngeneic corneal transplantation of C57BL/6 mice, also by performing flow cytometry of corneal graft homogenates. Finally, because A1AR stimulation increases transforming growth factor beta (TGFB), we then tested the potential pro-inflammatory role of this TGFB increase by carrying out EIU experiments on PHE-treated TSP-1 null mice, which exhibit a failure in TGFB activation. Results: A1AR stimulation with topical PHE induced dramatic exacerbations of EIU, as reflected by flow cytometry, histology, and bradford assay results. These exacerbated responses could be completely prevented by co-treatment with topical PRAZ, which also significantly alleviated EIU in mice not treated with PHE. Topical PHE also led to a significant increase in neutrophilic infiltration of syngeneic corneal grafts. TSP-1 null mice exhibited similar EIU responses by comparison with wildtype mice, and did not experience detectable exacerbations after PHE treatment. Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts Conclusions: Local A1AR stimulation exacerbates acute ocular inflammation via an effect that appears to involve TGFB expression, and function. Commercial Relationships: Paola A. Durand, None; Yaohong Tan, None; Darakshan Fatmi, None; Xiaomei Xia, None; Egla Suarez, None; Victor L. Perez, None; Jose L. Vega, None Support: None Program Number: 6236 Poster Board Number: D1096 Presentation Time: 8:30 AM - 10:15 AM Effect Of P2Y2 Deficiency On Experimental Autoimmune Uveitis Development Laure E. Caspers1, Lia J. Relvas1, Remi Dewispelaere1, Maya Makhoul2, Didier Communi2, Jean-Marie Boeynaems2, Bernard Robaye2, Catherine Bruyns2, François Willermain1. 1Ophthalmology, Univ of Brussels-St Pierre Hosp, Brussels, Belgium; 2Univ of Brussels-IRIBHM, Brussels, Belgium. Purpose: To study the potential role of the nucleotide receptor P2Y2 in the development of experimental autoimmune uveitis (EAU). Methods: EAU were induced in WT and P2Y2 KO mice by direct immunization with IRBP peptide 1-20 or adoptive transfer of in vitro restimulated semi-purified lymphocytes from spleen and draining lymph nodes isolated from immunized mice. Clinical and histological scores were used to grade disease severity. Lymphocyte proliferation was measured by thymidine incorporation and cytokine secretion by ELISA. Flow cytometry was used to analyze cell surface marker expression. Results: EAU clinical scores were slightly decreased in KO mice. Similarly, adoptive transfer of semi-purified lymphocytes from KO into C57Bl/6 WT mice induced significantly less disease than the transfer of WT cells into C57Bl/6 WT animals. Analysis of spleen and lymph node cells of immunized animals showed no differences in term of cell populations. However, in vitro restimulation of KO cell induced less proliferation and cytokine (IFN gamma, TNF alpha and IL-17) secretion as compared to WT cells. Conclusions: Our data show that P2Y2 mice are less susceptible to develop an autoimmune response against IRBP peptide 1-20, influencing the development of EAU. Those data are consistent with the hypothesis that P2Y2 play a role of danger signal receptors. Commercial Relationships: Laure E. Caspers, None; Lia J. Relvas, None; Remi Dewispelaere, None; Maya Makhoul, None; Didier Communi, None; Jean-Marie Boeynaems, None; Bernard Robaye, None; Catherine Bruyns, None; François Willermain, None Support: None Program Number: 6237 Poster Board Number: D1097 Presentation Time: 8:30 AM - 10:15 AM Role of iC3b-CR3 interaction in Experimental Autoimmune Anterior Uveitis Bharati Matta, Purushottam Jha, Puran S. Bora, Nalini S. Bora. Ophthalmology, Jones Eye Institute-UAMS, Little Rock, AR. Purpose: To explore the role of interaction between complement activation product - iC3b and its receptor CR3 in experimental autoimmune anterior uveitis (EAAU). Methods: Male Lewis rats (5-6 weeks old) were immunized with bovine melanin associated antigen (MAA) emulsified (1:1) in complete Freund’s adjuvant (CFA) in the hind footpad. Animals were sacrificed on different days post-immunization and lymphocytes were purified from the popliteal lymph nodes (LNs) using histopaque gradient. Lymphocytes were stained with anti-CD4 APC and anti-iC3b followed by staining with FITC labeled secondary antibody (Ab). In a separate set of experiment, cells were stained with anti-CD11b/c PE, anti-CD3-APC, antiCD45RA-FITC and anti-iC3b followed by staining with PerCP labeled secondary Ab. CFSE labeled lymphocytes purified from the popliteal LNs on day 5 postimmunization were plated in serum free CellGroR Medium containing rat iC3b in the presence of different concentration of anti-CD11b and anti-iC3b Abs separately for 5 days in the presence of bovine MAA (20 µg/ml). On day 6 non-adherent cells were collected and stained with anti-CD4-APC. The proliferation data was collected using FACS Calibur and was analyzed using Win MDI. Results: During the induction of EAAU, iC3b is deposited on antigen presenting cells (APCs) and CD4+ T cells present in the popliteal LNs. Out of total CD4+ T cells, the percentage of CD4+ T cells that are positive for iC3b started to increase as early as day 2, peaked on day 7 post-immunization and started to decrease after that. Out of total CD11b/c+ CD3- CD45RA- cells, the percentage of cells that are iC3b positive sharply increased at day 2 and peaked on day 7 post-immunization. Treatment with anti-CD11b and anti-iC3b Abs separately inhibited the proliferation of CD4+ T cells in response to MAA in a dose dependent manner. Conclusions: Our results demonstrate that during the induction phase of EAAU, iC3b is deposited on CD4+ T cells and CD11b/c+ CD3- CD45RA- APCs that are present in the popliteal LNs of MAA sensitized Lewis rats. Our data further suggest that the interaction between iC3b and its receptor CR3 modulates CD4+ T cells responses in EAAU. Commercial Relationships: Bharati Matta, None; Purushottam Jha, None; Puran S. Bora, None; Nalini S. Bora, None Support: NIH grant EY018812 and the grant from Pat and Willard Walker Eye Research Center, Jones Eye Institute, University of Arkansas for Medical Sciences, Little Rock, AR. Program Number: 6238 Poster Board Number: D1098 Presentation Time: 8:30 AM - 10:15 AM Temporal Expression of miR-155 Correlates with the initiation and Development of Experimental Autoimmune Uveitis (EAU) Bernadette Marrero, Yu Chen-Rong, Chandrasekharam Nagineni, Charles Egwuagu. Immunology, NEI, Bethesda, MD. Purpose: MicroRNAs are small non-coding genes that regulate diverse cellular processes in vertebrates. Aberrant or enhanced expression of several miRNAs including miR-146, miR-21, Let-7e and miR-155 are associated with several pathogenic conditions of humans. For example, miR-155 has recently been implicated in the pathogenesis of CNS inflammatory diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). In this study, we have investigated potential involvement of miR-155 in intraocular inflammatory disease or uveitis. Methods: To investigate the potential involvement of miR-155 in ocular inflammatory diseases, we induced experimental autoimmune uveitis (EAU) in C57BL/6 mice by active immunization with IRBP in CFA. Severity and progression in EAU were assessed by fundoscopy and histopathology. miRNA expression in human retinal cells (ARPE-19), as well as, in the retina of mice with EAU was analyzed by real-time quantitative PCR. Results: Analysis of inflammatory cells in the retina during EAU revealed a 5.3fold increase of miR-155 expression in the retina compared to un-immunized mice. The increase in miR-155 expression correlated temporally with increase in proinflammatory cytokines and progression of EAU. In addition, culturing human RPE cells in medium containing pro-inflammatory cytokines induced an even a more dramatic increase in miR-155 compared to untreated cells. Conclusions: Dramatic increase of miR-155 in the retina during ocular inflammation and following exposure of ocular cells to inflammatory environment was dramatic and suggest that miR-155 may be implicated in mechanisms that promote of T cell-dependent tissue inflammation. Our data further suggest that miR-155 might be a promising therapeutic target for the treatment of uveitis and other autoinflammatory disorders. Commercial Relationships: Bernadette Marrero, None; Yu Chen-Rong, None; Chandrasekharam Nagineni, None; Charles Egwuagu, None Support: None Program Number: 6239 Poster Board Number: D1099 Presentation Time: 8:30 AM - 10:15 AM DAP-12, a Major Immunomediator, Either Promotes or Suppresses EAU Development Barbara P. Vistica1, Vanessa Montalvo-Reddin1, Guangpu Shi1, Lindsey Nugent1, Laura Quigley2, Daniel W. McVicar2, Igal Gery1. 1Lab of Immunology, National Eye Institute, Bethesda, MD; 2Cancer and Inflammation Program, NCI-Frederick, Frederick, MD. Purpose: DAP12 (DNAX-activating protein of 12kDa), a transmembrane signaling molecule, plays a major role in the immune response by transducing a variety of activation signals from receptors on antigen-presenting and other leukocytes. The role of DAP12 in the etiology of pathogenic autoimmunity has been controversial, since DAP12 deficient mice reportedly exhibit either enhanced or depressed experimental autoimmune diseases. Here, we compared DAP12 deficient mice and wild type (WT) controls for their susceptibility to EAU induction and immune responsiveness to the uveitogenic antigen Methods: DAP12 deficient mice and their C57Bl/6 WT controls were obtained from two animal facilities, on the same campus, both specific pathogen free (SPF) but with different standards for helicobacter and other organisms. The mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) and their development of EAU was evaluated by fundoscopy and histological examination. Lymphoid cells from draining lymph nodes were cultured with IRBP to measure proliferation and cytokine release. Results: Although disease in control mice from both facilities was comparable DAP12 deficient mice from the SPF facility with less stringent health standards were superior to their WT controls in both the severity of their ocular inflammation and the levels of proliferation and of IL-17 and interferon gamma produced by their lymphocytes. In contrast, DAP12 deficient mice from the cleaner rodent barrier facility were inferior to their controls by both parameters. Conclusions: Our data provide a possible resolution to the controversy concerning the role of DAP12 in the induction of EAU: deficiency in this molecule can either promote or suppress the pathogenic response, even when the same experimental system is used. The difference we observed between the two portions of the study could be attributed only to conditions within the animal facilities that shape the immune systems. Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts Commercial Relationships: Barbara P. Vistica, None; Vanessa MontalvoReddin, None; Guangpu Shi, None; Lindsey Nugent, None; Laura Quigley, None; Daniel W. McVicar, None; Igal Gery, None Support: None Program Number: 6240 Poster Board Number: D1100 Presentation Time: 8:30 AM - 10:15 AM Inhibition of CdK5 Attenuates Experimental Autoimmune Uveitis Zili Zhang1, Xiumei Wu1, Jie Duan1, James T. Rosenbaum2. 1Pediatrics, Oregon Health & Science University, Portland, OR; 2Ophthalmology, Casey Eye InstituteOHSU, Portland, OR. Purpose: T cells are essential for the development of autoimmune uveitis. Our previous study showed that OX40, a key co-stimulatory molecule, plays a pivotal role in enhancing T cell response and ocular inflammation. However, the molecular mechanism by which OX40 regulates uveitogenic T cells remains to be fully elucidated. Recently, cyclin dependent kinase 5 (CdK5), a unique serine/threonine kinase, has been implicated in T cell activation. Furthermore, CdK5 exerts several key downstream effects in common with OX40. Thus, we sought to determine whether CdK5 is a critical intermediary of OX40 signaling, leading to exacerbation of uveitis. Methods: The splenocytes of DO11.10 mice were activated by cognate antigen ovalbumin (OVA) for up to 72 hours. Flow cytometry was performed to compare intracellular CdK5 expression between CD4+OX40+ and OX40- T cells. In addition, these cells were treated with CdK5 inhibitor Roscovitine, and T cell apoptosis was measured by Annexin V staining and MitoCapture assay. Lastly, experimental autoimmune uveitis (EAU) was induced by immunization of B10.RIII mice with interphotoreceptor binding protein. Some mice were further treated with OX40 activating antibody, and EAU score was determined on day 21 to assess the effect of Roscovitine on OX40-enhanced uveitis. Results: To explore the relationship of OX40 with CdK5, we categorized DO11.10 CD4+ cells to 2 distinct populations based on surface OX40 expression. After OVA activation, OX40+ cells expressed more intracellular CdK5 than OX40lymphocytes. In addition, blocking phosphatidylinositol 3-kinases, which mediates OX40 signaling pathway, reduced the expression of CdK5. Moreover, inhibition of CdK5 by Roscovitine led to the decrease of activated CD4+CD44+ lymphocyte number, which coincided with increased T cell apoptosis. Finally, weekly treatment of Roscovitine attenuated the severity of OX40-enhanced EAU. Conclusions: These results implicated CdK5 in OX40-augmented T cell response and suggest that targeting CdK5 may be a novel therapeutic strategy for treating T cell-mediated uveitis. Commercial Relationships: Zili Zhang, None; Xiumei Wu, None; Jie Duan, None; James T. Rosenbaum, None Support: NIH grant EY016788 Program Number: 6241 Poster Board Number: D1101 Presentation Time: 8:30 AM - 10:15 AM Immunological Inhibition of Pigment Epithelium-Derived Factor (PEDF) ? Charles E. Thirkill. Ocular Immunology Research Lab 1220 Surge III, UC Davis, Davis 95616, CA. Purpose: The biological activity of Pigment Epithelium-Derived Factor (PEDF, molecular weight 45 kd) includes the inhibition of the activity of Vascular Endothelial Growth Factor (VEGF,molecular weight 45 kd with smaller isoforms). Both of these ubiquitous factors are multi-functional. Of relevance to this study is the recognized expression of PEDF and VEGF within the retinal pigment epithelium. The combination of these two factors normally contributes to the balance of activity that maintains ocular homeostasis. Any immunological inhibition of PEDF might result in an imbalance encouraging the blood vessel propagation typical of several diseases that involve uncontrolled vascular proliferation, such as that seen in some cancers, diabetic retinopathies and AgeRelated Macular Degenerations. A search for evidence that might implicate the immunological inhibition of PEDF was performed on cancer patients presenting with evidence of the 45 kd CAR syndrome. Rationale: PEDF shares the same molecular mass as the unidentified 45 kd CAR antigen raising suspicion they might be one and the same molecule. The possibility of immunological inhibition of the activity of PDEF might then be of significance in both tumor growth, and its role in maintaining introcular equilibrium. The option that the 45 kd CAR antigen may be the 45 kd glycoprotein VEGF was included in these inquiries, but the potent antibody activity against this antigen did not support the likelihood. Methods: Western blot analyses were performed on the patient’s antibody activity with the protein components of pig retina, and in vitro cultivated retinal pigment epithelium (ATCC ARPE-19 CRL-2302). The 45 kd component of retinal pigment epithelium was immunoprecipitated from an extract of cultured RPE using 45 kd reactive cancer patient’s serum, and the precipitate subjected to proteomic analysis. Results: Preliminary proteomic analyses implicate pigment epithelium-derived factor as the 45 kd antigen of interest. Conclusions: The possibility of an antibody-mediated interferece in the biolocal activity of pigment epithelium-derived factor leads to a testable hypothesis: “Immunological inhibition of the modulating anti-vascular proliferation properties of pigment epithelium-derived factor results in a loss of homeostasis leading to the excessive production of blood vessels that typifies some forms of cancer, and retinopathies that involve uncontrolled vascular propagation”. If this hypothesis proves correct it will introduce the prospect of ameliorating the pathological process of unwanted vascular spread through approriate targeted immunomodulation therapy. Commercial Relationships: Charles E. Thirkill, Athena Diagnostics (P) Support: RPB and NEI core grant 1P30 EY12576-7 Program Number: 6242 Poster Board Number: D1102 Presentation Time: 8:30 AM - 10:15 AM Label-free LC-MSMS-based Differential Proteome Analysis of Vitreous from Autoimmune Uveitis Cases Stefanie M. Hauck1, Florian Hofmaier2, Johannes Dietter1,3, Marcel Blindert1, Elisabeth Kremmer4, Margarete E. Swadzba2, Barbara Amann2, Cornelia A. Deeg2, Marius Ueffing1,3. 1Department of Protein Science, Helmholtz Center Munich, Neuherberg, Germany; 2Department for Veterinary Sciences, Institute of Animal Physiology, Munich, Germany; 3Centre for Ophthalmology, Institute for Ophthalmic Research, Tubingen, Germany; 4Institute for Molecular Immunology, Helmholtz Center Munich, Munich, Germany. Purpose: Equine recurrent uveitis (ERU) is a devastating immune mediated inflammation targeting the inner eye and ultimately leading to blindness in the affected animals. The widespread disease (10%) is characterized by recurring episodes of inflammation followed by quiescent stages. ERU represents the only spontaneous animal model for human autoimmune uveitis and thus serves as a valuable model for exploration of as yet unknown pathomechanisms relevant for disease progression and relapses. Since vitreous is directly adjacent to the inflamed retina this compartment facilitates indirect exploration of ongoing disease related events in the retina. To investigate protein expression changes related to the disease, we undertook a comprehensive differential proteome profiling of ERU and healthy vitreous. Methods: Proteins from vitreous samples of healthy horses and ERU cases (n=16) were subjected to tryptic digestion and analysed by liquid chromatography mass spectrometry (LC-MSMS; OrbiTrap). All detected peptide features were aligned and statistically analysed for differential abundance based on cumulated peak intensities. Peptides were identified by database search (Ensembl, Equus Caballus) and resulting protein IDs were grouped according to their mode of regulation. Pathway enrichment analyses (Consensus PathDB and Genomatix Pathway Systems) were used for identification of overrepresented pathways per condition. Candidate proteins were validated by western blots on a large sample collection. Results: Highly sensitive comparisons of vitreous samples resulted in identification of 251 proteins, 105 of these were identified with very high confidence (2 or more peptides, p<0.01). 26 proteins were found upregulated in ERU cases, while 44 were downregulated. Pathway enrichment analyses with these regulated proteins resulted in the identification of several significantly overrepresented pathways, among them “ECM-receptor interaction” (KEGG) and “Wnt signaling” (INOH). From these pathways, selected candidates were validated by western blots and mode of regulation observed by quantitative mass spectrometry was confirmed in all cases. Interaction network analyses based in literature mining enabled to suggest potential pathomechanisms and will be discussed in detail. Conclusions: Quantitative label-free proteomics based on LC-MSMS identified a signature of protein markers in the vitreous of uveitic eyes that point to dynamic changes in both extracellular matrix homeostasis and wnt signalling. As both molecular networks are mechanistically linked to inflammation as well as vascular changes in the eye, members of these protein networks are likely involved in the pathogenesis of uveitis. Commercial Relationships: Stefanie M. Hauck, None; Florian Hofmaier, None; Johannes Dietter, None; Marcel Blindert, None; Elisabeth Kremmer, None; Margarete E. Swadzba, None; Barbara Amann, None; Cornelia A. Deeg, None; Marius Ueffing, None Support: German Federal Ministry of Education and Research: HOPE - FKZ 01GM0852; SFB 571 A5 Deeg and DFG De 719/2-2. Program Number: 6243 Poster Board Number: D1103 Presentation Time: 8:30 AM - 10:15 AM Hsa-mir-let 7i Treatment Suppresses Tlr4 Mediated Mitochondrial Oxidative Stress In Giant Cell Arteritis Narsing A. Rao, Sindhu Saraswathy. Ophthalmology, Doheny Eye Institute, Los Angeles, CA. Purpose: Our previous findings have shown downregulation of 15 miRNAs related to TLR4, its signaling molecules, and mitochondrial oxidative stress in giant cell arteritis (GCA). One of the 15 miRNAs, hsa-let-7i is validated to target TLR4. In this study, we investigate the role of hsa-let-7i in the modulation of TLR4 and mitochondrial oxidative stress in GCA that lead to amplification of the inflammatory process. Methods: Non-GCA temporal artery biopsy specimens were exposed to LPS and Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts subjected to total RNA isolation and immunohistochemical studies to detect TLR4, CD209 (dendritic cells) and CD31 (endothelial cells). qPCR analysis was performed to detect TLR4 and CD83 gene expression. miRNA PCR Array (SABiosciences, Frederick, MD) was performed using specific primers for hsa-mirlet 7i, mir-let 7a, mir-10a, mir-106b, mir-221. Another group of similar specimens were treated with miRNA hsa-let-7i mimic and LPS. Scrambled miRNA and samples without LPS treatment were used as controls. TLR4 expression was studied by qPCR analysis. Another set of specimens were exposed to LPS and then treated with the TLR4 inhibitor OXPAPC or miRNA hsa-let-7i mimic or scrambled microRNA. DNA damage marker 8-OHdG was colocalized with the mitochondrial marker COX IV by immunohistochemistry. Results: TLR4 was localized in the dendritic cells in the adventitia region of the temporal artery after LPS exposure whereas it was absent on the endothelial layer. miRNA-hsa-let 7i in LPS treated temporal artery specimens were significantly downregulated compared to non-treated controls.TLR4 was markedly upregulated in the temporal artery exposed to LPS. Such overexpression was suppressed with miRNA hsa-let-7i mimic treatment whereas it was not altered with scrambled microRNA treatment. 8-OHdG was colocalized with COX-IV in the adventitia of the temporal artery exposed to LPS. Such damage was absent in the artery treated with the TLR4 inhibitor or with miRNA hsa-let-7i mimic. Conclusions: The increased TLR4 expression on the dendritic cells of adventitia indicates that the initial inflammatory insult in GCA could begin in the adventitia. This study reveals that TLR4 activation from LPS leads to mitochondrial oxidative stress, and such TLR4 mediated damage can be prevented by the miRNA hsa-let-7i mimic treatment. Commercial Relationships: Narsing A. Rao, None; Sindhu Saraswathy, None Support: NIH grants: EY017347, EY019506, EY03040 and RPB Program Number: 6244 Poster Board Number: D1104 Presentation Time: 8:30 AM - 10:15 AM Amelioration of Experimental Autoimmune Uveoretinitis by Inhibiton of Toxic AGEs Formation Zhenyu Dong1A,1B, Nobuyoshi Kitaichi2,1B, Daiju Iwata1A,1B, Ryo Ando1A,1B, Junichi Fukuhara1A,1B, Anton M. Lennikov1A,1B, Atsuhiro Kanda1A,1B, Kousuke Noda1A,1B, Shigeaki Ohno1C, Susumu Ishida1A,1B. ADepartment of Ophthalmology, BLaboratory of Ocular Cell Biology and Visual Science, CDepartment of Ocular Inflammation and Immunology, 1Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2Department of Ophthalmology, Health Sciences University of Hokkaido, Sapporo, Japan. Purpose: Advanced glycation end products (AGEs) are permanently modified protein derivatives, forming through non-enzymatic glycation of amino groups. Since glyceraldehyde-derived AGEs have strong toxicity, it is considered as toxic AGEs (TAGE). TAGE has been demonstrated to play an important role in the pathogenesis of chronic inflammatory diseases through interaction with its receptor for AGEs (RAGE). Pyridoxamine, one of the vitamin B6 derivatives, has been demonstrated to inhibit the Amadori rearrangement in the process of AGEs production. In this study, we quantified the serum TAGE levels in patients with endogenous uveitis. In addition, therapeutic effects of pyridoxamine were examined on experimental autoimmune uveoretinitis (EAU), a murine model of human endogenous uveitis. Methods: One hundred three consecutive patients of uveitis (31 VKH disease patients in convalescent stage, 21 with HLA-B27 associated uveitis, 14 with Behcet disease, and 37 with sarcoidosis) and 33 healthy volunteers were enrolled. Serum TAGE levels of participants were quantified with ELISA. In EAU experiments, female B10.BR (H-2k) mice were immunized with interphotoreceptor retinoidbinding protein (IRBP)-derived peptide emulsified with complete Freund’s adjuvant containing M.tuberculosis, and continuously administered 200mg/kg or 400mg/kg of pyridoxamine every 24 hours orally. Severity of EAU was evaluated both clinically and histopathologically. Translocation of NF-κB p65 into nucleus, and retinal levels of TAGE were also examined immunohistochemically. Results: Serum TAGE levels of uveitis patients with each etiology were significantly higher than those of healthy controls (P<0.001 for all). EAU was significantly milder both clinically and histopathologically when treated with 400mg/kg of pyridoxamine (P<0.05). TAGE levels were decreased in both sera and retina (P<0.05 for both), and translocation of NF-κB p65 into nucleus was downregulated in retinas in pyridoxamine-treated EAU mice. Conclusions: TAGE levels were upregulated in sera of uveitis patients, and systemic administration of pyridoxamine ameliorated EAU. Our results suggest that TAGE-RAGE system contributes to the phathogenesis of uvetitis and is a novel therapeutic target for endogenous uveitis. Commercial Relationships: Zhenyu Dong, None; Nobuyoshi Kitaichi, None; Daiju Iwata, None; Ryo Ando, None; Junichi Fukuhara, None; Anton M. Lennikov, None; Atsuhiro Kanda, None; Kousuke Noda, None; Shigeaki Ohno, None; Susumu Ishida, None Support: None Program Number: 6245 Poster Board Number: D1105 Presentation Time: 8:30 AM - 10:15 AM Ophthalmological and Histological Manifestations of IgG4-Related Disease Elise Philippakis, Valerie Touitou, Coralie Bloch-Queyrat, Aude Rigolet, Frederic Charlotte, Phuc LeHoang, Bahram Bodaghi. Pitie Salpetriere Hospital, Paris, France. Purpose: IgG4-related disease is defined by both elevated serum IgG4 over 135mg/dL and histopathological features, such as lymphocytes and IgG4+ plasma cell infiltration (ratio IgG4+ plasma cells/IgG+ plasma cells over 40%), associated with typical tissue fibrosis or sclerosis. The aim of this study is to determine the ophthalmological and histocytological features of IgG4-related disease. Methods: Patients with biopsy-proven hyper-IgG4 syndrome, seen between 2009 and 2011 in a single tertiary center, were retrospectively reviewed. Clinical features were analyzed, and compared with histological features and with patient's prognosis. Results: Three patients were included in the study. Mean age at diagnosis was 58 years (range:50-63 years). One patient presented with unilateral painful propotosis, associated with third and fifth cranial nerves palsies. One patient had recurrent anterior scleritis, and one patient presented with chronic conjonctival infiltration. None of them had extra-ocular involvement (FDG-PET-scan). Other inflammatory conditions were ruled out. Histocytology demonstrated lymphoplasmocytosic proliferation with overexpression of IgG4+ plasma cells and signs of fibrosis. All patients responded to corticosteroids. However one patient developped orbital fibrosis and required the adjunction of an immusuppressive drug. Conclusions: IgG4 related disease is a new and rare entity, which can be challenging to diagnose when clinical features are atypical , such as isolated conjonctivitis, scleritis and orbital infiltrations. Histopathology is the gold-standard for the diagnosis. Prognosis is determined by fibrosis, sclerosis and systemic involvement(pancreatitis, retroperitoneal fibrosis and hepatitis).Treatment is not consensual, but is based on corticosteroids, associated most of the time with immunosuppressants in case of fibrosis or cortico- dependant lesions. Commercial Relationships: Elise Philippakis, None; Valerie Touitou, None; Coralie Bloch-Queyrat, None; Aude Rigolet, None; Frederic Charlotte, None; Phuc LeHoang, None; Bahram Bodaghi, None Support: None Program Number: 6246 Poster Board Number: D1106 Presentation Time: 8:30 AM - 10:15 AM Ocular Immune Pathological Analysis in a Murine Model of Anterior Scleritis Hiroko Taniguchi1, MingCong Wang1, Atsuo Nakajima2, Junko Hori1. 1 Ophthalmology, Nippon Medical School, Tokyo, Japan; 2Rheumatology, Tokyo Metropolitan Police Hospital, Tokyo, Japan. Purpose: As the sclera comprises type II collagen, scleritis is often associated with autoimmune diseases such as rheumatoid arthritis. We have previously reported the establishment of an anterior scleritis model through modification of a collageninduced arthritis model. The present study conducted ocular immunological analysis in this same model to investigate the scleritis pathology. Methods: Arthritis and anterior scleritis were induced in 8-week-old DBA/1J mice via subcutaneous injection of bovine type II collagen emulsified with complete Freund’s adjuvant into the posterior neck (primary immunization) and 21 days later into the periocular region (secondary immunization). Eyeballs were excised at 3, 5 and 8 weeks after secondary immunization and analyzed histologically and immunohistologically. Results: Clinical findings comprised severe arthritis and dilation of scleral blood vessels. Anterior scleral thickening was observed histologically, with significantly more inflammatory infiltrating cells in the anterior sclera compared to control mice. Infiltration of CD4+ and CD11b+ cells was observed, but no CD11c+ cells were present, while deposition of plasma cells (CD138), complement (C3) and immunoglobulin (IgG and IgM) was particularly evident in the region of the anterior sclera in contact with the ciliary body. Conclusions: T cells, macrophages, plasma cells, complement and immunoglobulins were present in the sclera of the collagen-induced anterior scleritis model, suggesting the involvement of immunocomplex deposition in the induced scleritis of the present model. Commercial Relationships: Hiroko Taniguchi, None; MingCong Wang, None; Atsuo Nakajima, None; Junko Hori, None Support: Grants-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science Program Number: 6247 Poster Board Number: D1107 Presentation Time: 8:30 AM - 10:15 AM Erythrocyte Sedimentation Rate and C-Reactive Protein in Anterior Uveitis Justin D. Marsh, Bethany B. Markowitz. University of South Carolina, Columbia, SC. Purpose: Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are laboratory tests used for monitoring disease processes due to their utility as nonspecific markers of inflammation. Not infrequently, systemic inflammatory processes show evidence of anterior segment inflammation. This study aimed to Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts evaluate ESR and CRP values during periods of anterior uveitis to determine if ocular inflammation was sufficient to elevate these laboratory values to levels considered outside the normal range. Methods: A retrospective study was performed by reviewing patient charts with ICD-9 diagnosis codes of anterior uveitis (364.01, 364.02, 364.04, 364.3) seen at University of South Carolina Ophthalmology Specialty Clinics over a two year period. From within this subset, charts were further reviewed to select patients in which there was a recorded ESR or CRP value within seven days of documented anterior uveitis by an attending ophthalmologist or resident within the department. Sixteen patients were found that met these criteria, all of which had ESR values within one week of examination. Five of the sixteen patients also had CRP values documented within one week of examination. Additional data including age, sex, degree of inflammation, etiology of inflammation, and bilaterality were recorded as documented. In cases of bilateral anterior uveitis, the degree of inflammation was determined by the more severe eye. ESR and CRP values were then stratified based on reference range values. ESR was considered within the normal range when < 20 mm/hr for patients of age < 40. For patients of age > 40, the normal reference range was calculated as < Age/2 for males, and < (Age +10)/2 for females. CRP was considered within the normal reference range when < 10 mg/L regardless of age or sex. Results: Sixteen patients were evaluated, two of which had multiple examinations corresponding with laboratory data. ESR and CRP were found to be within the normal reference range in 57% (12/21) and 60% (3/5) of patients, respectively. In patients with laboratory data on the same day as examination, ESR and CRP were within the normal reference range in 55% (6/11) and 67% (2/3), respectively. In patients with at least 2+ cellular reaction documented, ESR was found to be within the normal reference range in 60% (6/10) of patients. Conclusions: While ESR and CRP may be elevated in multiple inflammatory processes, anterior uveitis alone does not appear to be sufficient to yield elevated values beyond the normal reference range. In this study, ESR and CRP were within the normal range in 57% and 60% of patients with anterior uveitis, respectively. Commercial Relationships: Justin D. Marsh, None; Bethany B. Markowitz, None Support: None Program Number: 6248 Poster Board Number: D1108 Presentation Time: 8:30 AM - 10:15 AM Antiphospholipid syndrome in Mexican population Claudia Recillas-Gispert, Juan Carlos Martinez. Ophthalmology, INCMNSZ, Mexico City, Mexico. Purpose: To determine the prevalence of Antiphospholipid syndrome in the Mexican population over 10 years, and the frequency of ocular manifestations, and which of those cases were the first manifestation of the disease. Methods: Retrospective, observational, transversal and descriptive study reviewing the medical files and records from all patients with Antiphospholipid syndrome’s diagnostic with descriptive statistical analysis for age, gender, type of antiphospholipid syndrome (primary or secondary), ocular manifestations and visual acuity at our hospital in a period of 10 years. Results: We include 332 patients with diagnostic of Antiphospholipid syndrome. Mean age at the moment of the diagnostic was 29.54 years. 278 were women (83.73%). 141 patients (42.85%) were diagnosed with Primary Antiphospholipid syndrome (PAPS), which 9 patients (6.38%) were ocular PAPS; 191 patients (57.17%) with Secondary Antiphospholipid syndrome (SAPS), 14 patients (7.33%) presented one ocular thrombotic episode without other systemic episode. Patients with ocular vasso-oclusive manifestations were 15 with central vein vascular oclussion (CRVO):8, central arteriy vascular oclusion (CAVO): 6, branch retinal artery oclusion: 1, optic neuritis:12, retinal vasculitis:7 amaurosis fugax:3. Other ocular autoimmune manifestations: epiescleritis: 2, escleritis:2, papiledema:1, internuclear ophthalmoplegic: 1, uveitis:1.Visual prognosis in patients with ocular manifestations presented a wide range associated to the type of alteration. Patients with legal blindness in PAPS were 3: 2 related to CRAO and the other one with optical neuritis, while patients with SAPS were 6: 2 with CRVO, 2 retinal vasculitis, 1 with CRAO and 1 with optic neuritis. Conclusions: Ocular manifestations in antiphospholipid syndrome have a low prevalence with a tendency to be more affected in the SAPS, may be because of comorbility with other diseases. However, they have great impact on the visual prognosis, so it is important to do timely assessment on suspected APS patients. Commercial Relationships: Claudia Recillas-Gispert, None; Juan Carlos Martinez, None Support: None Eye Research and Surgery Institution, Cambridge, MA; 3Ophthalmology, BayWiew Clinic, Mumbai, India; 4Ophthalmology, Tauber Eye Center, Kansas City, MO; 5Ophthalmology, Harvard Medical School, Boston, MA. Purpose: To evaluate the demographic characteristics, clinical features, ocular complications, and successful therapeutic regimens in patients with scleritis associated with inflammatory bowel disease (IBD). Methods: Retrospective case series. We reviewed the electronic health records of 500 patients with scleritis seen at two tertiary referral centers and selected the ones with IBD. Patient characteristics, clinical features, ocular complications and successful therapeutic regimens were evaluated. Results: Of 500 patients with scleritis, 11 patients had IBD (2.2%), 9 with Crohn´s disease (CD) and 2 with ulcerative colitis (UC); that includes 8 women and 3 men with a mean age of 47 years (range, 33 to 67 years). Six of the patients with CD had diffuse scleritis, 1 had nodular scleritis, 1 had necrotizing scleritis, and 1 had necrotizing scleritis and posterior scleritis; anterior uveitis was present in 2 patients, peripheral keratitis in 3 patients, and glaucoma in 1 patient but final visual acuity was not decreased in any patient. Scleritis occurred after several episodes of anterior uveitis in 2 patients. The 2 patients with UC had diffuse scleritis with anterior uveitis without corneal lesions or glaucoma or decrease of vision. There was no previous uveitis. Scleritis was the initial manifestation whose study led to the diagnosis of CD in 4 of the 9 patients and to the diagnosis of UC in all 2 patients. All 11 patients had arthritis and had recurrent scleritis. Successful therapeutic regimens in scleritis with CD included 1 patient with sulfasalazine, 1 patient with intravenous steroids, 3 patients with methotrexate, 2 patients with azathioprine, and 2 patients with infliximab. Successful therapeutic regimens in scleritis with UC included 1 patient with infliximab and 1 patient with sirolimus. Conclusions: Scleritis associated with IBD is more common in CD than in UC. It is usually recurrent and may take the form of any type of scleritis, including necrotizing and posterior scleritis. Although ocular complications may appear, they unfrequently cause decrease of vision. Patients who develop scleritis after recurrent anterior uveitis should be examined for CD. Scleritis associated with IBD is more common in patients with arthritis and may be the initial manifestation of CD or UC. Scleritis associated with IBD most often will require immunomodulatory therapy or biologic response modifier drugs. Commercial Relationships: Maite Sainz de la Maza, None; Nicolas Molina, None; Luis A. Gonzalez-Gonzalez, None; Priyanka P. Doctor, None; Joseph Tauber, None; Stephen Foster, None Support: None Program Number: 6250 Poster Board Number: D1110 Presentation Time: 8:30 AM - 10:15 AM Posterior Scleritis and Orbital Mass Associated to Positive Antineutrophil Cytoplasmic Autoantibodies Without Systemic Involvement MarÃa de los Angele Ramos Cadena1, Gustavo Aguilar Montes1, Matilde Ruiz Cruz2. 1Ophthalmology, Hospital General Dr. Manuel Gea Gonzalez, Mexico City, Mexico; 2Ophthalmology, Centro de Investigacion de Enfermedades Infecciosas del Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico. Purpose: To present a clinical case of a 16 year-old Mexican patient, without any relevant previous medical history and systemic symptoms, diagnosed with posterior scleritis associated to positive antineutrophil cytoplasmic autoantibodies (ANCA) directed to Proteinase 3 (PR3), with clinical involvement of the right eye. Methods: We performed a complete ophthalmologic examination, laboratory and immunologic tests, ocular ultrasound, orbit and paranasal sinus CT Scan and cranial MRI. Results: Funduscopic examination revealed papillitis, macular scar, serous retinal detachment, and vitritis. Ocular ultrasound of the right eye showed T-sign, enlargement of the posterior wall and intraorbital optic nerve. Laboratory results did not show renal or other systemic involvement. ANCA results showed positive cANCA, and Anti-PR3. CT Scan revealed a small mass in the orbit cone, close to the optic nerve entrance, and enlargement of the intraorbital portion of the optic nerve. MRI disclosed a normal intracraneal optic nerve. She was treated with intravenous methylprednisolone 1g per day, during three days, followed by oral prednisone at immunosuppressive dose, tapered according to evolution, discontinued after one month and a half. The ophthalmologic and imagenologic manifestations ceased after the first month of treatment. Conclusions: 50% of posterior scleritis are related to systemic involvement. This patient presented positive cANCA and Anti-PR3 even after corticosteroid treatment, which would usually imply systemic activity; but in this case the disease presented without extraocular manifestations. Immunosuppressive treatment should be indicated if systemic involvement occurs or ocular symptoms recur because of Program Number: 6249 Poster Board Number: D1109 Presentation Time: 8:30 AM - 10:15 AM Scleritis Associated with Inflammatory Bowel Disease Maite Sainz de la Maza1, Nicolas Molina1, Luis A. Gonzalez-Gonzalez2, Priyanka P. Doctor3, Joseph Tauber4, Stephen Foster2,5. 1Instituto Clinico Oftalmologia, Hospital Clinico Oftalmologia, Barcelona, Spain; 2Ophthalmology, Massachusetts Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts the high risk of vasculitic multiorgan affection and renal failure. Commercial Relationships: MarÃa de los Angele Ramos Cadena, None; Gustavo Aguilar Montes, None; Matilde Ruiz Cruz, None Support: None Program Number: 6251 Poster Board Number: D1111 Presentation Time: 8:30 AM - 10:15 AM Uveitis In Patients With Diabetes Mellitus Martha C. Fuentes, Cintia G. Sánchez-Balleza, Miguel Pedroza-Seres. Ocular Inflammation and immunology, CONVAL, Mexico, Mexico. Purpose: 1) Describe diabetes frequency in population with uveitis attending Conde de la Valenciana Institute, 2) describe demographic characteristics and 3) asses the relationship between hyperglycemia and inflammatory reaction in patients with diabetes and active uveitis. Methods: Prospective case series, observational and descriptive. We recruited patients with active uveitis and diabetes mellitus. The initial evaluation of patients was conducted by two researchers and included complete ophthalmic history, best corrected visual acuity (BCVA), slit lamp biomicroscopy and fundus evaluation. The degree of inflammatory activity was determined according to SUN. Central glucose was requested at the time of diagnosis of uveitis or reactivation and during every visit. Comparison of blood glucose and degree of inflammatory activity was performed for all the observation points. Glycemia was related to the degree of inflammatory activity using Spearman test. Results: 315 patients with uveitis were evaluated, 48 patients were diabetic (15.2%), two patients had type 1 diabetes (2%). We found 38 female (79.1%), unilateral uveitis presented in 27 cases (56.2%); mean age was 56.75, time course of diabetes was 10.64 years. The most frequent cuases of uveitis were nongranulomatous anterior uveitis 29.1% (n=14), panuveitis 12.5% (n=6), VKH 12.5% (n=6), herpetic uveitis 10.4% (n=5) and scleritis 10.4% (n=5). Twenty five (51.02%) patients presented its first episode of uveitis. There is a statistically significant positive correlation (p<0.05) between the anterior chamber inflammation and hyperglycemia in non granulomatous idiopatic anterior uveitis and herpetic uveitis. Conclusions: Non granulomatous anterior uveitis, VKH and panuveitis are the most frequent types of uveitis in patients with diabetes. There is a positive correlation between hyperglycemia and inflammation in anterior chamber in patients with anterior uveitis. The behavior of uveitis in these patients is more aggressive and occurs more often bilaterally. Commercial Relationships: Martha C. Fuentes, None; Cintia G. SánchezBalleza, None; Miguel Pedroza-Seres, None Support: None Program Number: 6252 Poster Board Number: D1112 Presentation Time: 8:30 AM - 10:15 AM Risk Factors Associated with the Relapse of Uveitis in Patients with Juvenile Idiopathic Arthritis Ujwala H. Baheti1, Alaa Radwan1, Cheryl Arcinue1, Ravi Parikh1, Ashik Mohamed2, C Stephen Foster1. 1Ophthalmology, Massachusetts Eye Research and Surgery Institution, Cambridge, MA; 2Hyderabad Eye Research Foundation, L V Prasad Eye Institute, Hyderabad, India. Purpose: To identify risk factors associated with relapse of uveitis in patients with juvenile idiopathic arthritis (JIA) - associated chronic or recurrent uveitis following treatment with immunomodulatory therapy (IMT) and a drug free remission of 1 year. Methods: Retrospective chart review of 30 patients with JIA associated uveitis, who were successfully treated with IMT to a state of corticosteroid-free remission, and subsequently remained in remission following discontinuation of IMT for a period of at least 1 year. In subsequent follow up, some patients had relapse of uveitis while others continued to be in remission. Remission was defined as <1+ cells in the anterior chamber or vitreous. Relapse was defined as > 1+ cells in the anterior chamber or vitreous. We compared patients in remission with those who relapsed, in an effort to evaluate risk factors associated with the relapse. Results: Out of 30 patients, 17 (56.7%) remained in remission while 13 (43.3%) relapsed. The patients in the remission group received IMT earlier (median of 9 months, inter-quartile range [IQR] 6-12 months) in the course of disease from diagnosis as compared to patients in the relapse group (median of 78 months, IQR 36-120 months) (Mann-Whitney test, p = 0.0004). Moreover, the patients in the remission group had received treatment with IMT at a younger age (median age 6 years, IQR 4-9 years) as compared to the relapse group (median age 11years, IQR 8-15 years) (Mann-Whitney test, p = 0.04). None of the other factors studied revealed significant association with the relapse of uveitis. Conclusions: Patients with uveitis associated with JIA treated with IMT earlier in the course of disease and at a younger age have less likelihood of relapse of the uveitis following discontinuation of the IMT after a 2 year period of quiescence on IMT. Commercial Relationships: Ujwala H. Baheti, None; Alaa Radwan, None; Cheryl Arcinue, None; Ravi Parikh, None; Ashik Mohamed, None; C Stephen Foster, None Support: None Program Number: 6253 Poster Board Number: D1113 Presentation Time: 8:30 AM - 10:15 AM Risk Factors for Loss of Visual Acuity among Patients with Uveitis Associated With Juvenile Idiopathic Arthritis: The SITE Study Jennifer E. Thorne1, Anthony Gregory1, Ebenezer Daniel2, C Stephen Foster3, Douglas A. Jabs4, Grace A. Levy-Clarke5, Robert B. Nussenblatt6, James T. Rosenbaum7A, Eric B. Suhler7B, John H. Kempen8. 1Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, MD; 2Ophthalmology, University of Pennsylvania, Philadelphia, PA; 3Ophthalmology, Ocular Immunol & Uveitis Fndtn, Cambridge, MA; 4Ophthalmology, Mount Sinai School of Medicine, New York, NY; 5Tampa Uveitis, Tampa, FL; 6National Eye Inst/NIH, Bethesda, MD; AOphthalmology, B Uveitis Clinic/Portland VAMC, 7Casey Eye Institute-OHSU, Portland, OR; 8 Ophthal-Biostatistics & Epidemiol, Scheie Eye Inst/Univ of Penn, Philadelphia, PA. Purpose: To describe the incidence of and risk factors for ocular complications and loss of visual acuity (VA) in patients with juvenile idiopathic arthritis (JIA)associated uveitis. Methods: 327 patients (599 affected eyes) with JIA-associated uveitis managed at five tertiary ocular inflammation clinics in the United States were identified from the Systemic Immunosuppressive Therapy for Eye Diseases (SITE) Cohort Study. Demographic and clinical characteristics were obtained for every eye of every patient at every visit via medical record review by trained expert reviewers. Loss of VA to 20/50 or worse and to 20/200 or worse and the development of ocular complications were assessed. Results: At presentation, 240 (40.3%) eyes had a VA of 20/50 or worse; 144 (24.2%) had a VA of 20/200 or worse; and 359 (60.2%) had at least one ocular complication. Over a median follow up of 3 years (range = 1 month to 25 years), the rates of VA loss to the 20/50 or worse and 20/200 or worse thresholds were 0.18 and 0.09 per eye-year (EY), respectively. Among the affected eyes, the incidence of developing at least one new ocular complication over follow up was 0.15/EY (95% confidence interval [CI]: 0.13/EY, 0.17/EY). However, among eyes with uveitis that had no complications at presentation, the rate of developing at least one ocular complication during follow up was 0.07/EY (95% CI: 0.04, 0.11). After controlling for confounding factors, presence of posterior synechiae, active uveitis, and prior intraocular surgery were statistically significantly associated with VA to the 20/50 or worse and 20/200 or worse thresholds at presentation and during follow up. Increasing anterior chamber cell was associated with increased rates of visual loss in a dose-dependent fashion. Use of immunosuppressive drugs reduced the risk of visual loss, particularly for the 20/50 or worse outcome (hazard ratio = 0.43, 95% CI: 0.23, 0.81, P<0.01). Conclusions: Ocular complications and vision loss were common in our cohort. Active uveitis had a dose-dependent association with vision loss and use of immunosuppressive drugs reduced the risk of vision loss; thus reinforcing the recommendation that aggressive control of intraocular inflammation is critical to the prevention of visual loss among patients with JIA-related uveitis. Commercial Relationships: Jennifer E. Thorne, None; Anthony Gregory, None; Ebenezer Daniel, None; C Stephen Foster, None; Douglas A. Jabs, None; Grace A. Levy-Clarke, None; Robert B. Nussenblatt, None; James T. Rosenbaum, None; Eric B. Suhler, None; John H. Kempen, None Support: NIH/NEI R01 EY014943, Research to Prevent Blindness, Mackall Foundation, NEI Intramural Funds, Dept of Veterans Affairs, KURE Fund Program Number: 6254 Poster Board Number: D1114 Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2012 Annual Meeting Abstracts Presentation Time: 8:30 AM - 10:15 AM In Search Of Intraocular Biomarkers In Uveitis Associated With Juvenile Idiopathic Arthritis (jia) Viera Kalinina Ayuso1A, Jolanda D. de Groot-Mijnes2, Jojanneke Dekkers1B, Lenneke de Visser1A, Aniki Rothova1A, Joke H. de Boer1A. AOphthalmology, B Virology, 1UMC Utrecht, Utrecht, The Netherlands; 2Virology, Univ Medical Center Utrecht, Utrecht, The Netherlands. Purpose: To investigate the presence of specific protein profiles in aqueous humour (AH) from patients with juvenile idiopathic arthritis (JIA) associated uveitis. Methods: Totally 116 children participated in this study. Aqueous humor (AH) (n=73) and serum samples (n=105) were collected. The samples were analyzed using surface-enhanced laser desorption/Ionisation time-of-flight (SELDI-TOF) technique. The samples were divided in the following 4 groups: definitive JIA; presumed JIA (not meeting the International League against Rheumatism criteria completely); other uveitis entities and non-inflammatory controls. Results: In the definitive JIA group significantly higher expression of five proteins was found compared with other uveitis entities and controls (6672; 8725; 13762; 22313; 33410 Da). Definitive JIA and presumed JIA samples showed comparable protein profiles in AH and did not differ in any of the peak cluster intensities. Expression of protein peaks in AH showed significant positive and negative bivariate correlations. Both definitive and presumed JIA samples could be distinguished from other uveitis entities and controls by the presence of 13762 Da protein and absence of 8255 Da protein in AH. Protein expression in AH of patients with JIA and suspect JIA uveitis was strongly associated with activity of inflammation. Conclusions: Our results suggest the presence of intraocular JIA-specific proteins which expression is correlated with activity of uveitis independently of definitive or presumed JIA status. This could indicate similar molecular intraocular processes in both groups of patients and support the hypothesis of an atypical manifestation of JIA with primary uveitis. Further identification of potential biomarkers should be performed. Commercial Relationships: Viera Kalinina Ayuso, None; Jolanda D. de Groot-Mijnes, None; Jojanneke Dekkers, None; Lenneke de Visser, None; Aniki Rothova, None; Joke H. de Boer, None Support: Dr. F.P. Fischer stichting, ODAS stichting, SNOO Program Number: 6255 Poster Board Number: D1115 Presentation Time: 8:30 AM - 10:15 AM Dramatic Effect Of Bolus Cyclophosphamide In Two Severe Cases Of Lupus Retinopathy Soydan Kurun, Andy Locatelli, Toufik Maalouf, Karine Angioi. Ophthalmology, Regional Univ Hosp Ctr Brabois, Nancy, France. Purpose: Reporting dramatic effect of bolus cyclophosphamide in two severe cases of lupus retinopathy occurred in young women. Methods: Two young women aged 22 and 31 with active systemic lupus erythematosus (SLE) consulted for decreased visual acuity (4/10 P4 OU, and 8/10 P2 OD, 1/20 P14 OS respectively). Results: In our two patients, fundus examination highlighted a bilateral macular oedema, numerous cotton wool spots, perimacular exudates, flame hemorrhages suggestive of the diagnosis of lupus retinopathy. Clinical and biological criteria allowed the diagnosis of severe SLE due to neurolupus in one case, and lupus glomerulonephritis in the other. Aggressive treatment based on steroids and cyclophosphamide cures was decided because of presence of both sight- threatening lesions and multi systemic involvement. Rapid improvement in systemic and biological signs of SLE was observed with parallel disappearance of retinal signs. Full recovery of visual acuity was obtained within 2 to 3 months in our two patients. Prevalence of SLE varies between 10 and 50 p 100,000. Presence of ophthalmological manifestations is estimated between 5 and 50% of the patients depending on the series, and the most frequent one is lupus retinopathy. Prompt treatment combining corticosteroids and cyclophophamide cures followed by mycophenolate mofetil enables regression of retinal signs and improvement in visual acuity, along with clinical and self- immune and inflammatory tests (C3, C4, Anti Nuclear Factor and Anti DNA Antibodies) Conclusions: In severe active forms of SLE an immediate specific treatment combining cyclophosphamide cures and corticosteroids is mandatory to obtain not only improvement of systemic manifestations but also complete visual recovery. Commercial Relationships: Soydan Kurun, None; Andy Locatelli, None; Toufik Maalouf, None; Karine Angioi, None Support: None Melissa Meyer zu Hörste1,2, Elena Stroeher1, Yang Zhang2, Katharina Roeck3, Jens Fischer3, Utta Bercher-Pfannschmidt1, Anja K. Eckstein1, Erich Gulbins2. 1 Department of Ophthalmology, University Hospital Duisburg-Essen, Essen, Germany; 2Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany; 3Institute of Pharmacology and Clinical Pharmacology, University of Dueseldorf, Duesseldorf, Germany. Purpose: To study mechanisms of pathologic proliferation of orbital fibroblasts and the transformation of these cells to adipocytes, excessive production of extracellular matrix, and inflammation which are hallmarks in the pathogenesis of Graves ophthalmopathy (GO). These processes finally result in remodeling of the orbital tissue with the typical symptoms of lid retraction, periorbital swelling, disfiguring proptosis, optic nerve compression, and impaired ocular motility caused by fibrotic changes in the extraocular muscles. Methods: Human orbital fibroblasts were obtained from 12 patients with active, severe GO and from 12 healthy control subjects. The cells were characterized by immunofluorescence assay and flow cytometry. Expression of acid sphingomyelinase (ASM) was determined by Western blot techniques, ASM activity was measured by degradation of [14C]-labeled sphingomyelin to [14C]labeled phosphorylcholine, and Ceramide-levels were detected by DAG kinase assay and immunofluorescence in orbital fibroblasts. Cell proliferation was determined by BrdU-incorporation, hyaluronan (HA) production was assessed by a HA-binding protein based assay, intracellular reactive oxygen species (ROS) were determined by the dichlorofluorescein assay, and cell viability was measured by the MTT-assay. Results: ASM activity was inhibited with amitryptiline and with siRNA technology. We demonstrate that a stable change in the phenotype of orbital fibroblasts from GO patients results in a constitutive overactivity of ASM and a high release of ceramide in these cells. High activity of the acid sphingomyelinase/ceramide system in GO orbital fibroblasts results in a constitutive increase of proliferation, release of reactive oxygen species, and formation of hyaluronan. These hallmarks of GO are corrected upon pharmacologic or siRNA-mediated inhibition of acid sphingomyelinase activity. Conclusions: Our findings identify the acid sphingomyelinase/ceramide system as a key mechanism in the pathophysiological changes affecting orbital fibroblasts during GO and provide a rationale for inhibiting acid sphingomyelinase activity as a novel treatment for GO. Commercial Relationships: Melissa Meyer zu Hörste, None; Elena Stroeher, None; Yang Zhang, None; Katharina Roeck, None; Jens Fischer, None; Utta Bercher-Pfannschmidt, None; Anja K. Eckstein, None; Erich Gulbins, None Support: IFORES grant Nr. D/D/107-40170 Program Number: 6256 Poster Board Number: D1116 Presentation Time: 8:30 AM - 10:15 AM Inhibition Of The Acid Sphingomyelinase/ceramide System Prevents Hallmarks Of Graves Ophthalmopathy Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected].