Immunology/Microbiology (IM)

85 Pages·2012·1.92 MB·English

by  Michele Appley

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Immunology/Microbiology (IM)

145 The Immunobiology of AMD eyes by quantitative RT-PCR.
Sunday, May 6, 2012, 3:15 PM - 5:00 PM Results: The continuous monitoring of the fundus for 2 months showed a slower
Hall B/C Poster Session progression or alleviation of retinal lesions in the treated right eyes as compared to
Program #/Board # Range: 1227-1238/D1056-D1067 the untreated left eyes. Among 23 pairs of eyes, 78.3% were improved, 8.7%
Organizing Section: Immunology/Microbiology stayed the same and 13.0% remained progressing in the lesion levels. Histology
Contributing Section(s): Retina+,Retinal Cell Biology confirmed the clinical observation. Even though there was no difference in the level
of A2E between the treated and the untreated control eyes, microarray analysis of
92 immune genes showed that IL-17a relative expression was substantially
Program Number: 1227 Poster Board Number: D1056
decreased after the treatment. The TNF-α expression showed a similar pattern of
Presentation Time: 3:15 PM - 5:00 PM
IL-17a. The results were consistent in duplicated arrays and confirmed by
Treatment With Recombinant Interleukin-17A Reduces ARPE-19 Cell Viability
quantitative RT-PCR.
Daniel Ardeljan, Yujuan Wang, De Fen Shen, Jingsheng Tuo, Chi-Chao Chan.
Conclusions: The study showed that intravitreous administration of recombinant
Immunopathology Section/Laboratory of Immunology, National Eye Institute/NIH,
TSG-6 might stabilize retinal lesions in Ccl2-/-/Cx3cr1-/- mice on rd8 background.
Bethesda, MD.
Modulation of ocular immunological gene expressions, especially cytokine IL-17a,
Purpose: Age-related macular degeneration (AMD) is a degenerative disease that
could be one of the mechanisms.
impairs primarily photoreceptors and the retinal pigment epithelium (RPE) in the
Commercial Relationships: Chi-Chao Chan, None; Xiaoguang Cao,
macula. Recently, expression of significantly higher IL-17A transcripts was found
None; Defen Shen, None; Yujuan Wang, None; Jun Zhang, None; Joo Y. Oh,
in human AMD maculae and elevated serum IL-17A levels were measured in
None; Darwin J. Prockop, None; Jingsheng Tuo, None
AMD patients. IL-17A is known to induce a robust immune response, but its exact
Support: NEI Intramural Research Program
effect on the RPE remains elusive. This study explores the role of IL-17A in human
RPE cells.
Methods: Cultured ARPE-19 cells were serum-starved for 24h and then treated for Program Number: 1229 Poster Board Number: D1058
48h with recombinant IL-17A (100 ng/ml). A Taqman gene expression array of 92 Presentation Time: 3:15 PM - 5:00 PM
human signal transduction genes revealed IL-8 to be differentially expressed after Inflammatory Cytokine Induced VEGF-A and VEGF-C Secretion by Human
the treatment, which was further examined by quantitative RT-PCR. An experiment Retinal Pigment Epithelial Cells is Inhibited by the Polyphenolic Nutraceutical
to characterize the role of NF-kB, the upstream regulator of IL-8, was conducted by Resveratrol
co-incubating IL-17A and an NF-kB inhibitor (SN50, 18μM) with ARPE-19 cells. Chandra N. Nagineni1, Barbara Detrick2, John J. Hooks1. 1Lab of Immunology,
Apoptosis was evaluated by immunohistochemical detection of Caspase-3 and National Eye Inst/NIH, Bethesda, MD; 2Department of Pathology, Johns Hopkins
Caspase-9 activation and by flow cytometry using Annexin V/7AAD. An MTT University, Baltimore, MD.
assay was performed to evaluate ARPE-19 cell viability with respect to changes in Purpose: Vascular Endothelial Growth Factors (VEGFs) play critical roles in
IL-17A doses. choroidal neovascularization (CNV) in age-related macular degeneration (AMD).
Results: IL-17A-treated ARPE-19 cells had substantially increased IL-8 mRNA Retinal pigment epithelium (RPE) and choroid are the key tissues in the
along with elevated Caspase-9 activity and diminished Caspase-3 activity as pathogenesis of AMD. We have recently reported that inflammatory cytokines
detected by immunohistochemistry. Inhibition of NF-kB with SN50 resulted in (ICM) upregulate VEGFs expression by human RPE cells (HRPE). Resveratrol
increased apoptotic activity as measured by flow cytometry and this effect was (RSV), a phytoalexin polyphenol found in grapes and several plant products, was
marginally strengthened with the addition of IL-17A. The MTT assay revealed that shown to have beneficial effects against many age-related diseases,but its
IL-17A doses ranging from 1 pg/ml to 10 ug/ml decreased cell viability from usefulness for AMD is not known. In this study, we investigated the possible
approximately 20-35%. actions of RSV in regulating the expression of VEGFs by HRPE.
Conclusions: IL-17A might play a role in the apoptosis of RPE cells and IL-8 Methods: Primary cultures of HRPE were prepared from eyes from aged donors
might be one of its major mediators. IL-17A-treated ARPE-19 cells exhibit altered obtained from Eye Banks. Confluent cultures grown in 24 well plates were treated
caspase activity in a process largely but not completely mediated by the NF-kB with ICM (IFN-γ, 100 u /ml + TNF-α, 10 ng/ml + IL-1β, 10 ng/ml [10x conc.]) at
pathway. IL-17A was shown to have a negative effect on cell viability in culture. It different concentrations (0.2x, 1x, 5x and 10x) in the presence of RSV (0 - 50 uM)
seems that IL-17A is not only a mediator of inflammation, but also a regulator of for 24 h. HRPE cell viability was assessed using CellTiter 96 AQuoeous One
apoptosis. reagent. Levels of VEGF-A, VEGF-C, pigment epithelial derived factor (PEDF)
Commercial Relationships: Daniel Ardeljan, None; Yujuan Wang, None; De and endostatin in the culture supernatants were determined by ELISA. VEGF
Fen Shen, None; Jingsheng Tuo, None; Chi-Chao Chan, None mRNA expression was evaluated by RT-PCR. Effects of RSV on HRPE cell
Support: NIH Intramural Fund migration and proliferation were studied by in vitro wound closure assay.
Results: HRPE cell viability was not significantly affected by RSV and RSV+ICM
treatment for 24 hr. VEGF-A and VEGF-C secretion were significantly enhanced
Program Number: 1228 Poster Board Number: D1057
in HRPE cells by ICM even at very low (1x and 0.2x) concentrations. VEGF-A and
Presentation Time: 3:15 PM - 5:00 PM
VEGF-C secretion that was enhanced by ICM at a wide range of concentrations
Anti-inflammatory Recombinant TSG-6 Stabilizes The Progression Of Focal
(0.2x-10x) was inhibited significantly by RSV in a dose (10 -50 uM) dependent
Retinal Degeneration In A Murine AMD Model
Chi-Chao Chan1, Xiaoguang Cao1,2, Defen Shen1, Yujuan Wang1,3, Jun Zhang1, Joo manner. There were no significant effects of ICM and /or RSV on the secretion of
Y. Oh4, Darwin J. Prockop5, Jingsheng Tuo6. 1Immunopath Sect, Lab of Immunol, two anti-angiogenic molecules, PEDF and endostatin by HRPE cells. These data
National Eye Institute/NIH, Bethesda, MD; 2Ophthalmology, People's Hospital, demonstrate that RSV is a potent suppressor of both VEGF-A and VEGF-C
Peking University/Beijing, China; 3Zhongshan Ophthalmic Center, Sun Yat-sen secretion elevated during inflammatory conditions. RSV exhibited dose (0-50 uM)
University, China; 4Institute for Regenerative Medicine, Texas A&M Health dependent inhibition of wound closure in confluent HRPE cultures.
Science Center, Temple, TX; 5Immunopath Sect, Lab of Immunol, 2Institute for Conclusions: Our results suggest that RSV inhibits expression of VEGFs in HRPE
cells during inflammatory conditions, which are known to be associated with CNV
Regenerative Medicine, Texas A&M Health Science Center, Temple, TX;
6Laboratory of Immunology, National Eye Institute/NIH, Rockville, MD. in AMD. RSV also inhibited HRPE cell migration and /or proliferation. Thus, RSV
or its derivatives have great therapeutic potential as nutraceutical in controlling
Purpose: The inflammatory responses are detected in the retina of age-related
macular degeneration (AMD) patients and Ccl2-/-/Cx3cr1-/- mice on rd8 choroidal and /or retinal neovascularization in AMD.
Commercial Relationships: Chandra N. Nagineni, None; Barbara Detrick,
background, a model that develops progressive AMD-like retinal lesions including
None; John J. Hooks, None
focal photoreceptor degeneration, abnormal retinal pigment epithelium, and A2E
Support: Intramural program, NEI, NIH
accumulation. Tumor necrosis factor-inducible gene 6 protein (TSG-6) is an anti-
inflammatory protein and has shown to improve myocardial infarction and
chemically injured cornea in mice by suppressing inflammation. In this study, we Program Number: 1230 Poster Board Number: D1059
evaluated the effect of intravitreous injection of recombinant TSG-6 on the retinal Presentation Time: 3:15 PM - 5:00 PM
lesions of these Ccl2-/-/Cx3cr1-/- mice. CCL2/CCR2 Chemokine Axis Mediates Inflammatory Monocytes
Methods: Recombinant TSG-6 (400 ng) was intravitreously injected into the right Recruitment To Lesions Of Atrophic Age-related Macular Degeneration And
eyes of 2-month-old Ccl2-/-/Cx3cr1-/- mice. The left eyes were injected with PBS as Retinal Degeneration In Cx3cr1 Deficient Mice
controls. Funduscopic pictures were taken before injection and sequentially once a Florian Sennlaub1,2, Constance Auvynet3, Xavier Guillonneau1, Serge Camelo1,
month after injection. The mice were sacrificed 2 months after injection. Ocular Sophie Lavalette1, Jean-Louis Bourges2, Francine Behar-Cohen2, William Raoul1,
histopathology was examined. Retinal A2E, a major component of lipofuscin, was Christoph Combadiere3. 1Institut de la Vision, UMRS 986, University Pierre et
measured by HPLC. The microarray of ocular mRNA of 92 immunological genes Marie Curie, Paris, France; 2Ophthalmology, Hotel Dieu, Paris, France;
was performed. The genes showing differentiated expression in microarray were 3Laboratoire d'Immunologie Cellulaire, Inserm UMRS 945, Paris, France.
further compared between the injected right eyes and the contralateral (control)

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 by Scientific Section/Group – Immunology/Microbiology (IM)

Purpose: The chemokine/chemokine receptor axis CCL2/CCR2 has been shown to Minzhong Yu1A, Weilin Zou1B, Neal S. Peachey1A,2, Thomas McIntyre1B, Jinbo Liu1B.
mediate inflammatory monocyte recruitment in a number of neurodegenerative ACole Eye Institute, BDepartment of Cell Biology, Lerner Research Institute,
disorders. Intraocular CCL2 levels are increased in wet AMD. We have shown that 1Cleveland Clinic Foundation, Cleveland, OH; 2Research Service, Louis Stokes VA
subretinal macrophages/microglial cells (Mφ/MCs) accumulation in Cx3cr1-/- mice Medical Center, Cleveland, OH.
is associated with photoreceptor degeneration. We here investigated intraocular Purpose: The observations that single nucleotide polymorphisms of complement
CCL2 expression and CCR2+ inflammatory monocyte recruitment in Geographic alternative pathway components are associated with the risk of AMD development
Atrophy (GA) and Cx3cr1-/- mice and their influence on photoreceptor indicate that complement plays an important role in AMD pathology. How these
degeneration in Cx3cr1-/- mice. variants lead to photoreceptor degeneration is not known. To better understand the
Methods: [CCL2] was measured by ELISA in aqueous humors of 18 GA patients relationship between complement activation and the retina, we examined mice
and 22 age-matched control patients with no signs of AMD undergoing cataract lacking the receptors for complement activation fragments C3a (C3aR-/-), C5a
surgery. CCL2 expression and the number of CCR2+inflammatory monocytes were (C5aR-/-) or both (C3aR-/-/C5aR-/-).
analyzed in macular sections of donor tissues with GA lesions (n=8) and age Methods: Complement mutant mice (C3aR-/-; C5aR-/-; C3aR-/-/C5aR-/-) were
matched control eyes (n=5). CCL2 expression (rt-PCR and ELISA) and studied along with WT littermates from ages of 6 weeks up to 14 months. After
inflammatory monocyte infiltration (cytometrie) was analyzed in light-injured overnight dark adaptation, strobe flash ERGs were used to examine outer retinal
Ccl2-/-, Cx3cr1-/- and Ccl2-/-Cx3cr1-/- mice. The influence of inflammatory function and a dc-ERG technique was used to measures ERG components
monocyte infiltration on subretinal Mφ/MC accumulation and photoreceptor generated by the retinal pigment epithelium (RPE). Retinas were examined by
degeneration was analyzed using clodronate liposomes and CCR2 inhibitors. immunohistochemistry and microscope.
Results: We show that atrophic age-related macular disease (AMD) is associated Results: C3aR was localized to the RPE and all cell layers of the neural retina;
with increased intraocular CCL2 levels and subretinal CCR2+ “inflammatory” C5aR expression was restricted to the RPE and ganglion cell layer. We observed a
monocyte infiltration. Using the Cx3cr1-/- mouse model of subretinal Mφ/MC progressive decline of ERG a- and b-wave amplitudes in C3aR-/- and C3aR-/-/C5aR-
accumulation and photoreceptor degeneration, we show that pharmacological /-mice, which was correlated with cellular losses in the ONL and INL. These
inhibition of CCR2, genetic deletion of CCL2, and depletion of circulating changes were not observed in C5aR-/- animals. Amplitude reductions were,
monocytes prevents inflammatory monocyte recruitment, MC/Mφ accumulation, however, noted in dc-ERGs recorded from C5aR-/- mice.
and photoreceptor degeneration in vivo. Our data suggests that CCL2/CCR2 Conclusions: Genetic deletion of receptors for the complement activation
inhibition represents a powerful tool for controlling inflammation and fragments C3a and C5a leads to cell-specific defects that match the cellular
neurodegeneration in AMD. localization of these receptors in the WT retina. These indicate that receptor
Conclusions: Our data suggests that CCL2/CCR2 inhibition represents a powerful signaling through these pathways contribute to retinal cell survival.
tool for controlling subretinal inflammation and neurodegeneration in AMD. Commercial Relationships: Minzhong Yu, None; Weilin Zou, None; Neal S.
Commercial Relationships: Florian Sennlaub, None; Constance Auvynet, Peachey, None; Thomas McIntyre, None; Jinbo Liu, None
None; Xavier Guillonneau, None; Serge Camelo, None; Sophie Lavalette, Support: AHAF Award M2008-063, Foundation Fighting Blindness Center Grant;
None; Jean-Louis Bourges, None; Francine Behar-Cohen, None; William Research to Prevent Blindness; VA Medical Research Service.
Raoul, None; Christoph Combadiere, None
Support: ERC-2007 St.G. 210345; ANR- 08-MNPS -003 LSHB-CT-2005-518167
Program Number: 1233 Poster Board Number: D1062
Presentation Time: 3:15 PM - 5:00 PM
Program Number: 1231 Poster Board Number: D1060 Knockout Of Membrane-associated Complement Inhibitors Cd55 And Cd59
Presentation Time: 3:15 PM - 5:00 PM Reduces Retinal Light Damage
γδTcells Promote Experimental Choroidal Neovascularization In An Il-23- Delu Song1,2, Yafeng Li1, Imran Mohammed3, Ying Song1, Majda Hadziahmetovic1,
independent Manner Wenchao Song3, Joshua L. Dunaief1. 1Department of Ophthalmology, University of
Eiichi Hasegawa1, Takashi Shichita2, Yuji Oshima1, Atsunobu Takeda1, Takeru Pennsylvania, Scheie Eye Institute, Philadelphia, PA; 2Department of
Yoshimura1, Koh-hei Sonoda3, Tatsuro Ishibashi1, Akihiko Yoshimura2. 1Kyushu Ophthalmology, Chinese Academy of Medical Sciences & Peking Union Medical
Univ Grad School of Med, Fukuoka, Japan; 2Microbiology and Immunology, Keio College, Peking Union Medical College Hospital, China; 3Department of
Univ School of Med, Tokyo, Japan; 3Ophthalmology, Yamaguchi University, Ube, Pharmacology, University of Pennsylvania, Institute for Translational Medicine
Japan. and Therapeutics, Philadelphia, PA.
Purpose: Interleukin(IL)-17 is a proinflammatory cytokine associated with Purpose: To determine whether eliminating decay-accelerating factor
autoimmunity and defense against bacteria; it has been implicated in many (DAF/CD55) and CD59, two main membrane-associated complement regulatory
autoimmune diseases such as psoriasis, rheumatoid arthritis and uveitis. Previously, proteins, can protect the mouse retina against light-induced photoreceptor
we demonstrated that IL-17, which is mainly produced by γδTcells, promotes degeneration.
choroidal neovascularization (CNV) in mouse laser-induced model. Both IL-1β and Methods: CD55/CD59 double knockout mice (DKO) on a Balb/c background
IL-23 have been reported to be indispensable for IL-17 expression in Th17 cells, (mixed sex) and age/sex matched wild type mice were placed in constant bright
γδTcells and other IL-17 producing cells. Herein we report the expression of IL-1β white fluorescent light (10,000 lux) for 18 hours. Retinas were evaluated before
and IL-23 in laser-treated eyes and their involvement in IL-17 expression by and after light exposure. Retinal degeneration was assessed by histology 10 days
γδTcells. after exposure to damaging white light. Electroretinography (ERG) was used to
Methods: Adult wild-type C57BL/6J mice and IL-23p19 knockout (KO) mice evaluate retinal function. Complement factor H (CfH) and C3 levels in
received laser photocoagulation (wave length=532 nm, 0.1s, spot size=75 µm, neurosensory retina were assessed by qPCR and Western analysis.
power=200mW) to induce CNV. Cytokine expression in laser-treated eyes was Results: Light exposure resulted in substantial photoreceptor-specific cell death.
evaluated by quantitative real-time PCR at multiple time points. To analyze CNV Eliminating CD55 and CD59 protects the mice against light-induced photoreceptor
area, mice were perfused with fluorescein-lableled dextran on day 7. Eyes were degeneration. The retinal mRNA levels of Rhodopsin (Rho) in male CD55/CD59
harvested and fixed, and the choroid-scleral flatmount was subjected to image DKO mice were significantly higher than male WT at 10 days following light
analysis. FACS-sorted γδTcells were cultured with or without IL-1β or IL-23. After damage (P<0.001). The amplitude of both cone and rod b-waves were significant
48hrs, IL-17 expression in the culture supernatant was measured by ELISA. increased in DKO mice relative to WT mice. Morphological analysis demonstrated
Results: Both CNV area and IL-17 mRNA expression were comparable between CD55/CD59 DKO mice had a thicker ONL and better preserved photoreceptor
eyes from wild-type mice and IL-23p19KO mice. However, eyes from IL-23p19 inner and outer segments following 10 days of light injury. The retinal C3 protein
KO mice had significantly higher levels of IL-1β mRNA expression compared to level in no light damaged CD55/CD59 DKO mice was significantly lower than WT
eyes from wild-type mice. IL-17 production by cultured γδTcells was enhanced by mice without light challenge. Additionally, both the CfH protein level and mRNA
both IL-1β and IL-23, but it was also enhanced by IL-1β alone. level were significantly higher in DKO retinas compared to WT retina.
Conclusions: These results suggest that even without IL-23, IL-1β alone is Conclusions: Eliminating CD55 and CD59 systemically reduces the C3 levels in
sufficient to induce IL-17 production by γδTcells in the laser-treated eye. the neurosensory retina and protects photoreceptor from light-induced damage.
Commercial Relationships: Eiichi Hasegawa, None; Takashi Shichita, CfH was upregulated in CD55/CD59 DKO retinas and may have inhibited both the
None; Yuji Oshima, None; Atsunobu Takeda, None; Takeru Yoshimura, complement cascade and retinal damage induced by oxidized lipids.
None; Koh-hei Sonoda, None; Tatsuro Ishibashi, None; Akihiko Yoshimura, Commercial Relationships: Delu Song, None; Yafeng Li, None; Imran
None Mohammed, None; Ying Song, None; Majda Hadziahmetovic, None; Wenchao
Support: None Song, None; Joshua L. Dunaief, None
Support: ARVO Foundation for Eye Research
Program Number: 1232 Poster Board Number: D1061
Presentation Time: 3:15 PM - 5:00 PM
A Role For Complement Activation In Preventing Retinal Degeneration

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 by Scientific Section/Group – Immunology/Microbiology (IM)

Program Number: 1234 Poster Board Number: D1063 Support: Voltmann Stiftung, Akademie des Sehens
Presentation Time: 3:15 PM - 5:00 PM
Dysregulation Of Alternative Pathway Complement Components In Mice Program Number: 1236 Poster Board Number: D1065
Leads To An Increased Susceptibility To Tissue Damage Presentation Time: 3:15 PM - 5:00 PM
Susie E. Barker, Ulrich F. Luhmann, Jill A. Cowing, Scott J. Robbie, Alexander J. Local Anti-C5 Therapy Suppresses Experimental Choroidal
Smith, Robin R. Ali. Division of Molecular Therapy, UCL Institute of Neovascularization Through Reduction Of Macrophage Infiltrate
Ophthalmology, London, United Kingdom. Jian Liu1, David A. Copland1, Shintaro Horie1, B. Paul Morgan2, Lindsay B.
Purpose: Age-related macular degeneration (AMD) is the most common cause of Nicholson1, Andrew D. Dick1. 1Academic Unit of Ophthalmology, School of
late-onset blindness in the developed world. The importance of the complement Clinical Sciences, University of Bristol, Bristol, United Kingdom; 2School of
system in AMD pathogenesis is well established, and AMD-like symptoms are Medicine, Cardiff University, Cardiff, United Kingdom.
seen in patients with other complement dysregulation diseases. We investigated the Purpose: Increasing evidence demonstrates that abnormal complement activation
complement alternative pathway (AP) in wildtype, CFH+/- and CFH-/- mice to plays a critical role in the development of choroidal neovascularization (CNV), a
characterize how the lack of CFH changes the local and systemic activation of the complex pathological process underlying the wet form of age-related macular
AP components, and in response to acute retinal injury. degeneration (AMD). The present study aimed to determine the efficacy of local
Methods: Liver and whole eyes from C57Bl/6, CFH+/- and CFH-/- mice were administration of the murine anti-C5 mAb (BB5.1) to prevent cleavage of C5 and
analysed by western blot and quantitative RT-PCR to compare local versus suppress laser-induced CNV.
systemic production of C3,CFB, CFD, CFP and CFH. We performed serum C3a Methods: CNV was induced in C57BL/6 mice by laser photocoagulation (4 laser
ELISA to analyse the levels of activation of the complement cascade. The injury spots/eye). The BB5.1 mAb or an isotype control antibody (25μg/eye) were
response was tested by subretinal injection of PBS and eyes were assessed administered by intravitreal injection immediately following laser burn either as a
histologically for retinal pathology. single dose treatment, or a repeated regimen with an additional injection performed
Results: We observed a significant decrease in serum levels of CFH, correlating on day 7. The CNV formation was assessed by isolectin B4 staining on retinal
with the number of functional CFH alleles (p<0.05). As a consequence of CFH pigment epithelium (RPE)/choroid whole-mounts. Deposition of the end product of
depletion, C3 and CFB were significantly decreased consistent with the CFH complement activation, membrane attack complex (MAC), was detected by
genotype of the mice (p<0.05). There was no difference between CFH genotypes in immunostaining. Flow cytometric analysis and confocal microscopy were used to
the mRNA levels of C3, CFB, CFD or CFP in liver or eyes. There was no examine cell infiltrate in RPE/choroid and retina tissues. Expression of VEGF and
difference in whole C3 protein levels within liver or eye, but CFB was depleted in Arginase-1 (Arg-1) mRNA in the tissues were measured by real-time PCR.
the CFH-/- mice. C3a levels were significantly reduced in the serum of CFH+/- and -/- Results: Laser photocoagulation led to disruption of Bruch‟s membrane and RPE.
mice (p<0.05), but there was no significant difference between genotypes in C3a No significant new blood vessel growth was found at the laser-induced lesion sites
levels in liver or eye. Scoring of histological sections showed that CFH+/- and -/- before day 4, and fully-developed CNV was observed after day 7. During the early
mice had significantly higher levels of RPE damage following subretinal injection stage of CNV, there were elevated Arg-1 mRNA level and significant leukocyte
of PBS compared to wild types (p<0.05). infiltrate in the RPE/choroid, including accumulation of amoeboid macrophages
Conclusions: C3 and CFB are dysregulated in CFH deficient mice despite normal (CD11b+VEGF+) surrounding and within the lesions prior to formation of the new
levels of gene expression, indicating that the AP is continuously activated, leading blood vessels. Inhibition of C5 cleavage by local BB5.1 injection markedly
to increased degradation of C3 and CFB. CFH-/- mice show a greater injury suppressed MAC at the peak of MAC deposition (day 4). Furthermore, reduced
response to subretinal injection, suggesting a greater inflammatory reaction at the macrophage and neutrophil infiltration in the RPE/choroid were seen as early as 48
site of injection that is less controlled than in wild types due to the lack of CFH. hours following BB5.1 injection with diminished VEGF and Arg-1 mRNA
Therefore our data suggests that CFH protects tissues from damage that may be due expression in the RPE/choroid on day 4. Single dose administration showed a 52%
to lack of control of AP activation. reduction in CNV volume by day 7 compared to control treatment, but suppression
Commercial Relationships: Susie E. Barker, None; Ulrich F. Luhmann, of CNV was not maintained with blood vessel growth evident by day 14. However,
None; Jill A. Cowing, None; Scott J. Robbie, None; Alexander J. Smith, the repeated dose regimen of BB5.1 with treatments on days 0 and 7 significantly
None; Robin R. Ali, None reduced CNV volume by 56% at day 14.
Support: None Conclusions: This data demonstrates that blocking of C5 cleavage has clinical
potential to reduce CNV development via inhibiting complement activation, cell
Program Number: 1235 Poster Board Number: D1064 infiltrate and VEGF/Arg-1 expression. Repeated administration of anti-C5 mAb
Presentation Time: 3:15 PM - 5:00 PM may be required for effective suppression of the pathological disease, and would
Low Influence Of C5A On Retinal Pigment Epithelial Cells provide an alternative to or combined with current VEGF-targeting therapy for wet
Susanne Wasmuth1A, Katharina Lueck1A, Albrecht Lommatzsch1, Daniel AMD.
Pauleikhoff1. AOphtha-Lab, 1Augenaerzte at St. Franziskus Hospital, Muenster, Commercial Relationships: Jian Liu, None; David A. Copland, None; Shintaro
Germany. Horie, None; B. Paul Morgan, None; Lindsay B. Nicholson, None; Andrew D.
Purpose: Activated complement may be related to the development of age-related Dick, None
macular degeneration (AMD). C5a is an important anaphylatoxin and a side- Support: Dunhill Medical Trust and National Eye Research Centre (UK)
product during the activation of the complement cascade. This study addressed the
influence of C5a on retinal pigment epithelial (RPE) cells that play a key role in Program Number: 1237 Poster Board Number: D1066
AMD. Presentation Time: 3:15 PM - 5:00 PM
Methods: Human ARPE-19 cells were incubated with recombinant C5a in Carboxyethylpyrrole-specific T cells Promote M1 Macrophage Polarization:
different fetal calf serum (FCS) concentrations and were stained for C5b-9 using Innate and Adaptive Immunity Cooperate in the Early Onset of Age-Related
immunocytochemistry. The cell viability was assayed using thiazolyl blue. Macular Degeneration
Reactive oxygen species (ROS) were measured by conversion of nitro blue Fernando Cruz Guilloty1, Ali Saeed1, Stephanie Duffort1, Yaohong Tan1, Asha
tetrazolium. The content of vascular epithelial growth factor (VEGF) in the cell Ballmick1, Robert G. Salomon2, Victor L. Perez1. 1Ophthalmology, University of
culture supernatants was quantified by ELISA. The transepithelial resistance (TER) Miami, Miami, FL; 2Chemistry, Case Western Reserve, Miami, FL.
of ARPE-19 cells cultured on transwell inserts after apical and basal C5a addition Purpose: The immune system has emerged recently as a critical factor in the
was examined. pathogenesis of age-related macular degeneration (AMD), although precise cellular
Results: C5b-9 was not detected in response to C5a treatment. The viability of the and molecular mechanisms remain to be defined. Carboxyethylpyrrole (CEP) is an
RPE cells was only slighly influenced by C5a, even at high doses and prolonged oxidation fragment associated with AMD. Mice immunized with CEP-adducted
incubation. A slight increase in ROS was found, but the effect was negligible when mouse serum albumin (CEP-MSA) develop autoantibodies and AMD-like lesions,
compared to the influence of FCS. A constitutive VEGF production of the cells was providing an ideal setting to study immune responses that may be linked to AMD
measured. An enhanced secretion of VEGF was mostly dependent on the development. We have previously shown autoreactive T cell activation and a
incubation period. C5a exerted only minor effects. No clear TER alteration through tissue-specific role for macrophages in our model at the earliest stages of disease
C5a addition was observed. onset, prior to overt retinal damage. In this study we aimed to elucidate in more
Conclusions: Our previous findings after complement stimulation resulted in detail the potential cooperation of innate and adaptive immunity in AMD
induction of the membrane attacking complex C5b-9 and indicated various AMD- development.
like changes in RPE cells. In contrast, C5a alone had no significant influence on Methods: CEP-specific T cells from wild type C57BL/6 or BALB/c immunized
ARPE-19 cells. Although C5a has strong pro-inflammatory effects on other cell with CEP-MSA were stimulated and expanded ex vivo for 4 days. Bone marrow-
types, RPE cells might be more resistant and may need complete assembly of C5b- derived primary macrophages were generated from naïve or immunized mice. T
9. cells and macrophages were co-cultured at different ratios and different time points
Commercial Relationships: Susanne Wasmuth, None; Katharina Lueck, (6, 12, 24 and 48 hr), the supernatants were collected to measure cytokine
None; Albrecht Lommatzsch, None; Daniel Pauleikhoff, None

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 by Scientific Section/Group – Immunology/Microbiology (IM)

production by ELISA and RNA was isolated from each cell type to analyze gene 146 Immune Responses and Immunopathology
expression of relevant genes by quantitative real-time PCR. Macrophages were also Sunday, May 6, 2012, 3:15 PM - 5:00 PM
polarized toward either M1 or M2 phenotypes and injected subretinally, followed Hall B/C Poster Session
by eye harvest and gene expression analysis 7 days later. Program #/Board # Range: 1239-1255/D1068-D1084
Results: Macrophages become activated and polarized toward the M1 pathway (as Organizing Section: Immunology/Microbiology
measured by upregulation of TNF, IL-12, KC, IL-1b, and iNOS) when cultured in Contributing Section(s): Cornea,Retina+,Retinal Cell Biology
the presence of CEP-specific T cells, but only when the cognate antigen (CEP-
MSA) is included. Furthermore, there was also a downregulation of M2 markers,
Program Number: 1239 Poster Board Number: D1068
such as IL-10 and Arginase. Direct effects of local injection of macrophages within
Presentation Time: 3:15 PM - 5:00 PM
the retina in the context of CEP activation are currently under investigation.
Bioactive Sphingolipids As Mediators Of Ocular Inflammation: Observations
Conclusions: Our previous data provided strong evidence linking immune
In An Animal Model
responses against oxidative damage with early presence of macrophages at sites of Annie Y. Chan1, Robert Chen2, Donald S. Stone1, Annette Eckerd1, Nawajes
future AMD and may help to clarify the role of these cells in AMD pathology. We Mandal1. 1Ophthalmology, Oklahoma University Health Sciences Center,
now show a more direct effect of antigen-specific T cells on the polarization of M1 Oklahoma City, OK; 2Ophthalmology, Chengdu University, Chengdu City, China.
macrophages, which are associated with tissue damage. The exact nature of this
Purpose: To characterize and compare the clinical and histological response to
interaction in vivo is still unknown but is one of the main questions to be addressed
intravitreal (IV) ceramide (Cer) injection at varying doses over one month.
in future studies. We propose a two-step model for the onset of AMD: first, T cell
Methods: Varying doses of C-8 Cer were injected into rat eyes: 2ug (n=14), 10ug
activation leads to pro-inflammatory cytokine production (IFN-g and IL-17) that
(n=16), 20ug (n=14). Controls included IV injection of 100% DMSO (Cer vehicle,
primes macrophages for M1 polarization once they get recruited to the retina
n=9), BSS (n=9), needle puncture (n=7) or no intervention (n=3). Dihydroceramide
through chemokine signaling.
(DHCer, a non-bioactive sphingolipid) was also given IV at 2ug (n=2), 10ug (n=2),
Commercial Relationships: Fernando Cruz Guilloty, None; Ali Saeed,
and 20ug (n=2). Fundus photos, spectral domain (SD)-OCT, and fluorescein
None; Stephanie Duffort, None; Yaohong Tan, None; Asha Ballmick, None;
angiography (FA) were performed at 24 hr, 72 hr, 1 week, 2 weeks, and 4 weeks.
Robert G. Salomon, SKS Ocular (P); Victor L. Perez, SKS Ocular (C, P)
Additional eyes injected with similar doses were used for histologic comparison.
Support: FCG is a Howard Hughes Medical Institute Fellow of the Life Sciences
The degree of intravitreal cellular response, retinal edema or hemorrhage, vessel
Research Foundation, The Edward N. & Della L. Thome Foundation (VLP), NEI tortuosity, vitreous traction, and corneal changes (neovascularization or ulcers)
P30 EY014801, Research to Prevent Blindness. were documented. Retinal vessel tortuosity was graded on a scale of 0 (least)-3
(most), with a plus to indicate the presence of vessel dilation.
Program Number: 1238 Poster Board Number: D1067 Results: After 72 hours, 71% of the 2ug Cer group exhibited less than grade 2
Presentation Time: 3:15 PM - 5:00 PM vessel tortuosity, while 73.3% of the10ug and 64.3% of the 20ug groups showed
Vitamin D Reduce Retinal Inflammation And Clears Amyloid Beta grade 2 or greater tortuosity respectively. By 1 week, 66.7% of 2ug-injected eyes
Systemically In Aged Mice were less than grade 2, while 78.6% of 10ug-injected eyes and 71.4% of 20ug-
Vivian K. Lee, Jaimie Hoh Kam, Glen Jeffery. Institute of Ophthalmology, injected eyes were grade 2 or greater. At 2 weeks, 100% of 20ug-injected eyes were
University College London, London, United Kingdom. grade 2 or greater; over half were grade 3 or 3+. Optic nerve traction was present
Purpose: Vitamin D3 plays a key role in immune regulation and may protect only in 10- or 20ug-injected eyes, was progressive over time, and most severe in
against ageing. Excess amyloid beta deposition and inflammation have been known 20ug-injected eyes. OCT showed the greatest cellular reaction occurring within 72
as risk factors leading to age-related macular degeneration (AMD), the largest hours of injection and at higher doses of Cer. Low-grade inflammation that
cause of blindness in those over 50 years in developed countries. Vitamin D3 has resolved by 72hrs was observed in DMSO-injected eyes. Each remaining type of
been shown to significantly reduce retinal macrophage numbers, reduce control resulted in minimal observable inflammation on OCT or fundus
inflammation and clear amyloid beta in the eye and brain. These changes were photography. Histologic findings correlated with our imaging.
reflected in a significant improvement in visual function. Vitamin D3 has also been Conclusions: Sphingolipids such as Cer have recently been recognized as
linked to longer leukocyte telomeres, and epidemiologically to possible protection mediators of cellular processes including proliferation, migration, apoptosis, and
against AMD. Here, we examine the outer retina following vitamin D3 treatment to inflammation. The exact role of these mediators in human ocular diseases remains
specifically look at its inflammatory properties on outer segments. We also unexplored. Our data shows that IV injection of C-8 Cer, a bioactive sphingolipid,
examine its impact on amyloid beta in retinal and major systemic blood vessels in can generate a localized dose-dependent inflammatory response with an acute
aged mice. phase lasting up to 72 hours after exposure, followed by extensive vitreoretinal
Methods: Ultrastructure examination of the outer retina was analysed with fibrosis, especially around the optic nerve. Meanwhile, intravitreal injection of the
scanning electron microscopy (SEM). Cone density was estimated with peanut nonactive form of Cer, DHCer, resulted in virtually no inflammatory response.
agglutinin staining. Levels of proinflammatory cytokine, tumour necrosis factor-α Finally, we propose that C-8 Cer can be used as a potential model for investigating
(TNF-α) were also assessed using immunohistochemistry (IHC). Amyloid beta posterior uveitis, and neovascular retinopathies.
deposition was also studied using IHC in retinal and aortic blood vessels. Commercial Relationships: Annie Y. Chan, None; Robert Chen, None; Donald
Results: Aged vitamin D3 treated mice had markedly decreased deposition on their S. Stone, None; Annette Eckerd, None; Nawajes Mandal, None
photoreceptor outer segments as revealed in SEM images. These appeared clean Support: NIH/NCRR COBRE (RR17703); Research to Prevent Blindness Inc.
with a relatively regular alignment. However, a greater level of deposition was USA
present on controls and in the majority of cases they appeared to be less regular in
their alignment, although no quantitative measures of these were made. Together Program Number: 1240 Poster Board Number: D1069
with decreased deposition, the IHC revealed that levels of TNF-α, a pro- Presentation Time: 3:15 PM - 5:00 PM
inflammatory cytokine also were significantly reduced. Estimates of cone Immune Cell Infiltration into Cornea after Alkali Burn is modulated by
photoreceptor numbers did not reveal significant differences between the groups, Amniotic Membrane Transplantation in Mice
although numbers in vitamin D3 treated animals were slightly higher. Analysis of Nicholas J. Cutrufello1, Samantha Herretes2, Jose Echegaray3, Victor L. Perez4.
amyloid beta levels in blood vessels revealed reductions in the vitamin D3 treated 1Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami,
animals in both the retina and the aorta. FL; 2Ophthalmology, Bascom Palmer Eye Inst-Univ of Miami, Miami, FL; 3Dept
Conclusions: Vitamin D3 may have a range of therapeutic effects in its immune of Ophthalmology, University of Miami, Miami, FL; 4Ophthalmology, Bascom
protective role and its impact on inflammation may be significant for AMD. Palmer Eye Institute, Miami, FL.
Supplementation may be a route to reducing inflammation in aged human retina, Purpose: Corneal chemical burns are a common cause of potential vision loss.
delay photoreceptor degeneration and clear amyloid beta systemically. Alkaline agents, found in caustic soda lime, have both hydrophilic and lipophilic
Commercial Relationships: Vivian K. Lee, None; Jaimie Hoh Kam, properties allowing them to pass cell membranes easily making them particularly
None; Glen Jeffery, None damaging and able to gain access to the anterior chamber quicker. Hydroxyl ions
Support: None cause saponification of cell membranes, disrupt extracellular matrix, and ultimately
cell death. Alkali burns revoke the immune privilege of the cornea and may also
result in limbal stem cell deficiency. In this study we aim to visualize and assess
immune cell infiltration of the cornea after alkali insult and the immunomodulatory
affects of amniotic membrane transplantation (AMT).
Methods: C57 mice receive a corneal alkali burn of 0.5M NaOH for 20s and then
thoroughly flushed until a resulting corneal surface pH of 7-7.4 is reached. Mice
then receive AMT (AmbioDry2) with running 11-0 Nylon suture. Control groups
include alkali burn only, and alkali burn plus suture (no AMT). Mice were

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 by Scientific Section/Group – Immunology/Microbiology (IM)

clinically scored on clarity, vascularity, scar formtion, and fluorescein staining on inflammation.
post operation days (POD) 0, 1, 3, 5, 7, 10, 14, 21, 28. Eyes were then harvested Methods: SKH1-HrHr wild type mice and mice that express a constitutively active
and used for H & E staining and immunohistochemistry. Neutrophils, Stat6 (Stat6VT), V547A/T548A, (N=44, 24 respectively) are examined for the
macrophages, and B & T cells were stained for imaging and localization. Chimeric development of blepharitis. Eyelids and periorbital tissue were removed and
eGFP mice were also utilized to assess in vivo cell migration into the cornea after examined by H&E as well as Western blot analysis with antibodies against IL-4,
alkali burn. These eyes were scored using the methods above with the addition of Interferon γ, and CD3. Lid abnormalities of mutant and wild-type mice were
fluorescent imaging to visualize eGFP expressing cells. characterized. Real-time PCR analyses for Th1 and Th2 markers were performed
Results: Mice treated with amniotic membrane had lower corneal clarity scores on mRNA of both wild type and Stat6VT mice.
(clearer corneas; 1.7 vs. 3.15 at POD25) and had less corneal vascularity than Results: Compared to wild-type, Stat6VT transgenic mice exhibited increased
control and suture groups. Differences in groups began at POD10 and continued frequency of blepharitis and periorbital inflammation (N=44, P<0.05). The Stat6VT
through until the end of the experiment. Both control and AMT treated mice had transgenic mice demonstrated markedly increased eyelid thickness and elevated T-
high levels of neutrophil infiltration at POD3, with no significant difference. cell infiltrates (N=5, P<0.05). Kaplan-Meier analysis showed that approximately
However, AMT treated mice had fewer macrophages, B & T cells than mice in the 90% of the Stat6VT animals developed blepharitis as compared to none of the
alkali burn only group at POD 28. Less eGFP cells were also noted in AMT treated wild-type sibling controls over a 30-week period (N=44, 24 respectively). Real-
mice versus alkali burn only or suture/burn mice. time PCR analyses performed on the mRNA from both the wild type and Stat6VT
Conclusions: AMT is used in the treatment of many ocular surface disorders. In mice showed elevated levels of Th2 cytokines (Il-4, IL-10, and IL-13) and no Th1
this study we show the effect of AMT to decrease immune cell infiltration into the cytokine (IFN-γ) expression in the eyelid lesions isolated from the Stat6VT mice
cornea after alkali insult. (N=3, 4 respectively P<0.05).
Commercial Relationships: Nicholas J. Cutrufello, None; Samantha Herretes, Conclusions: We have identified a novel animal model of blepharitis implicating
None; Jose Echegaray, None; Victor L. Perez, None IL-4 and Stat6 signaling in the pathogenesis of chronic eyelid inflammation. The
Support: R01 EY018624-01, Research to Prevent Blindness Stat6VT transgenic mouse may be an excellent model system for examining future
medical therapeutics for immune-mediated blepharitis.
Program Number: 1241 Poster Board Number: D1070 Commercial Relationships: Callah West, None; Sonia DaSilva, None; Lou
Presentation Time: 3:15 PM - 5:00 PM Sun, None; Deena Mohamed, None; Jeffrey Travers, None; Yang Sun, None
Deletion of Thrombospondin (TSP)-1 in Dendritic Cells (DC) of the Support: AGS, Knights Templar
Conjunctiva Exacerbates Allergic Conjunctivitis (AC)
Daniel R. Saban, Sharmila Masli, Payal Khandelwal, Hyun S. Lee, Simona Program Number: 1243 Poster Board Number: D1072
Schelereth, Tomas Blanco. Ophthalmology, Schepens Eye Res Inst, MEE, Harvard Presentation Time: 3:15 PM - 5:00 PM
Medical School, Boston, MA. Amniotic Membrane And Limbal Mesenchymal Stromal Cells Possess Similar
Purpose: It is known that DCs contribute to an allergic reaction via activation of Immunosuppressor Properties
secondary T cell responses, though the molecular factors that regulate DCs in this Yonathan Garfias1A, Jessica Nieves1A, Mariana Garcia-Mejia1A, Carlos Estrada-
process are poorly understood. TSP-1 is an extracellular matrix protein that Reyes1A, Maria C. Jimenez-Martinez1B,2. AResearch Unit, BDept of Immunology,
modulates immunity and is abundantly expressed throughout the ocular surface, but Research Unit, 1Institute of Ophthalmology, Mexico City, Mexico; 2Biochemistry,
its role in allergy is unknown. We assessed whether TSP-1 influences the Faculty of Medicine, UNAM, Mexico City, Mexico.
contribution of DCs to allergic immunity in experimental AC. Purpose: Mesenchymal stromal cells (MSC) are non-hemopoietic cells with the
Methods: Wildtype (WT) vs. TSP1-/- mice were immunized with ovalbumin capacity to self-renew and differentiate into various cell lineages of mesenchymal
(OVA; 100µg)/ pertussis toxin (300ng)/ alum (1mg), and 14 d later mice were origin. Recently, the immune regulatory potential of MSC has been focused on. By
challenged with topical OVA eye drops (250 ug daily, 10 d consecutively). In other the one hand, amniotic membrane (AM) mesenchymal cells (AM-MSC) are
experiments, WT vs. TSP1-/- mice were adoptively transferred with primed WT T capable of inducing allogenic T cell suppression, as well as promoting a tolerogenic
cells (10^6), and then challenged. In a final experiment, TSP1-/- mice were microenvironment; by the other hand, mesenchymal stem cells obtained from
adoptively transferred with WT T cells, subconjunctivally (subconj) engrafted with limbal murine tissue possess immunomodulatory properties and inhibit
WT vs. TSP1-/- null bone marrow-derived (BM) DC (10^5), and then challenged. proinflammatory immune reactions. The aim of this study was to determine the
Masked clinical scoring was performed 20 min post challenge. Cytokines profiles immunosuppressive characteristics of two different mesenchymal populations
from in vitro recall assays were evaluated by ELISA, FACS enumeration of conj isolated from human AM and limbal tissues.
eosinophils (CD45+ Siglec F+), and serum OVA specific IgE were also assessed. Methods: Phenotypic analysis of AM-MSC and L-MSC were performed by means
Experiments (n>3 mice per group) were repeated at least twice. of molecular and flow cytometry assays. TCR stimulated peripheral blood
Results: Either in response to immunization or to adoptive transfer of WT T cells, mononuclear cells were co-cultured with either AM-MSC or L-MSC on a transwell
peak clinical scores were significantly (p<0.05) elevated (by ≥40%) in TSP1-/- system and T cell proliferation was documented by flow cytometry. ELISA method
mice relative to scores in WT controls. However, in TSP1-/- mice in response to was performed to detect IL-10 and TGFbeta immunosuppressive cytokines from
adoptively transferred WT T cells, such an increase in clinical scores as reversed by the culture supernatants.
subconj engraftment of WT but not TSP1-/- BMDCs. Likewise, this reversal was Results: Herein, we described that both AM- or L-MSC cell populations in vitro-
associated with a decrease in eosinophil recruitment (by 130%), a decrease expanded have similar phenotypic characteristics in comparison with those
(p<0.05) in IL-4 (by 2.3-fold), IL-5 (by 2.2-fold), and IL-13 (by 1.2-fold), and a described to bone marrow mesenchymal stromal cells. In addition, AM- or L-MSC
decrease (p<0.05) in OVA specific IgE levels (by 30%). co-cultured in a transwell system with peripheral blood mononuclear cells
Conclusions: AC is exaggerated by deletion of TSP-1 in immunized mice. This stimulated with anti-CD3/CD28 are capable to suppress TCR-engagement
effect is important during activation of secondary T cell responses, as exaggeration lymphocyte proliferation, which can be reverted using anti-TGFbetaRII
in TSP1-/- mice was observed even though WT T cells were adoptively transferred neutralizing antibodies. Moreover, TGFbeta1 was constitutively secreted by AM-
into these mice. Interestingly, however, subconj engraftment of WT DCs or L-MSC as determined by ELISA. Thus, TGFbeta1 secreted by Amniotic
completely reverses this exaggerated effect in TSP1-/- mice. Taken together, our Membrane as well as Limbal Mesenchymal Stromal Cells is able to suppress T cell
data suggest that TSP-1 expression by DCs regulates their capacity to mediate proliferation.
allergic immune responses in AC. Conclusions: Taken together these results, explain in part the immunosuppressive
Commercial Relationships: Daniel R. Saban, None; Sharmila Masli, features of these two cell populations obtained from two different human tissues.
None; Payal Khandelwal, None; Hyun S. Lee, None; Simona Schelereth, Commercial Relationships: Yonathan Garfias, None; Jessica Nieves,
None; Tomas Blanco, None None; Mariana Garcia-Mejia, None; Carlos Estrada-Reyes, None; Maria C.
Support: Lions Club of Massachusetts (DRS) Jimenez-Martinez, None
Support: "Conde de Valenciana" Foundation and CONACYT-SALUD-2011-1-
Program Number: 1242 Poster Board Number: D1071 160286
Presentation Time: 3:15 PM - 5:00 PM
Stat6 Mediated Blepharitis In An Atopic Dermatitis Model: Th2 Implication Program Number: 1244 Poster Board Number: D1073
In Chronic Inflammation? Presentation Time: 3:15 PM - 5:00 PM
Callah West1A, Sonia DaSilva1B, Lou Sun1C, Deena Mohamed1B, Jeffrey Travers1C, Expression of Inducible Nitric Oxide Synthase and Cationic Amino Acid
Yang Sun1A. AOphthalmology, BPathology, CDermatology, 1Indiana University, Transporter (CAT) In Endotoxin-Induced Uveitis
Indianapolis, IN. Chang-Hao Yang, Yi-An Lee, Chung-May Yang, Muh-Shy Chen. Ophthalmology,
Purpose: The purpose of this study is to characterize the eyelid inflammatory National Taiwan Univ Hospital, Taipei, Taiwan.
lesions found in the Stat6VT mouse, which is a murine model of atopic dermatitis. Purpose: Nitric oxide (NO) plays a significant role in the pathogenesis of ocular
We hypothesize that the constitutive activation of Stat6 leads to chronic periocular inflammation. The synthesis of NO depends on cell membrane protein cationic

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 by Scientific Section/Group – Immunology/Microbiology (IM)

amino acid transporter (CAT) to transfer arginine into the cell and inducible nitric marker expression was analyzed by immunofluorescence (CK-7 and MUC5AC,
oxide synthase (iNOS) to convert arginine into NO. The purpose of this study is to goblet cell; CK-4,stratified squmaous cell) and flow cytometry (MUC5AC).
examine the expression of CAT and iNOS in endotoxin-induced uveitis (EIU). Cultured goblet cells were treated for 24 hrs with 10 ng/ml of each of the
Methods: EIU was induced by a footpad injection of 200 μg LPS in Lewis rats. inflammatory cytokines previously detected in TSP-1 null inflamed conjunctiva
Proteasome inhibitor bortezomib (valcade) was injected intraperitoneally 30 (IFN-γ, TNF-α, IL-6, and IL-17). Cells were then stimulated with the cholinergic
minutes before the LPS administration. The rats were sacrificed 24 hours later and agonist carbachol for 1 hr and MUC5AC secretion evaluated by ELISA in the
the eyes were enucleated. The expression of iNOS and CAT mRNA and protein in supernatants of the treated cells.
the iris and ciliary body was determined by RT-PCT and Western blotting. The Results: Tear MUC5AC levels were significantly lower in TSP-1 null compared to
production of NO in aqueous was measured by ELISA. Immunochemical staining WT mice at both 8 and 12 weeks of age. Almost all cells in primary mouse goblet
of the iris and ciliary body was performed to evaluate the inhibitory effect of cell cultures expressed the goblet cell specific markers CK-7 and MUC5AC, but
valcade. . The interaction of NF-κB activation, CAT, and iNOS induction was were negative for the stratified squamous cell marker CK-4. Treatment with IFN-γ
assessed by electrophoretic mobility shift assay (EMSA). and TNF-α, significantly inhibited and IL-6 enhanced carbachol-mediated secretion
Results: Proteasome inhibitor valcade abrogated the inflammation of EIU as of MUC5AC. Exposure of goblet cells to IL-17 did not alter carbachol-mediated
demonstrated by immunochemical stain and the decreased NO production in MUC5AC secretion.
aqueous. The expression of iNOS and CAT mRNA and protein in EIU mouse eyes Conclusions: The pro-inflammatory cytokines expressed in the TSP-1 null mouse
were suppressed by valcade. EMSA revealed that NF-κB activation was conjunctiva have differential effects on the secretion of mucins produced by goblet
responsible for iNOS and CAT induction. cells in response to cholinergic stimulus resulting in altered tear quality. The loss of
Conclusions: NF-κB is an essential transcription factor not only for the induction ocular surface protection may further exacerbate ocular surface inflammation in
of iNOS, but also for the up-regulation of CAT. The simultaneous up-regulation of TSP-1 deficient mice.
CAT and iNOS may play an important role in the pathogenesis of EIU. Commercial Relationships: Laura Contreras-Ruiz, None; Arpita Gosh-Mitra,
Commercial Relationships: Chang-Hao Yang, None; Yi-An Lee, None; Bruce Turpie, None; Marie Shatos, None; Darlene A. Dartt,
None; Chung-May Yang, None; Muh-Shy Chen, None None; Sharmila Masli, None
Support: None Support: DoD grant W81XWH-10-1-0392
Program Number: 1245 Poster Board Number: D1074 Program Number: 1247 Poster Board Number: D1076
Presentation Time: 3:15 PM - 5:00 PM Presentation Time: 3:15 PM - 5:00 PM
Modulation of Eosinophil Responsiveness to TSLP-Mediated Degranulation Hmgb1, A Damage Associated Molecular Pattern Molecule, Is Critical For
by Conjunctival Epithelial Cells and Allergic Cytokines The Development Of T Cell-induced Autoimmune Uveitis
Ellen B. Cook1, James L. Stahl2A, Elizabeth D. Schwantes2B, Sameer A. Mathur2B, Guomin Jiang1, Henry J. Kaplan1, Huan Yang2, Deming Sun3, Amir Reza
Neal P. Barney2A. 1Ophthalmology and Visual Sciences, University of Wisconsin- Hajrasouliha1, Hui Shao1. 1Department of Ophthalmology, University of
Madison, Madison, WI; AOphthalmology and Visual Sciences, BMedicine, 2Univ of Louisville, Louisville, KY; 2The Feinstein Institute for Medical Research,
Wisconsin-Madison, Madison, WI. Manhasset, NY; 3Doheny Eye Institute, University of Southern California, Los
Purpose: Eosinophils and thymic stromal lymphopoietin (TSLP) are increased on Angeles, CA.
the ocular surface during allergic inflammation. Our previous studies demonstrated Purpose: To determine the role of HMGB1 (high mobility group box protein-1), a
that eosinophils respond to high concentrations of TSLP with degranulation. The major damage associated molecular pattern (DAMP), in ocular autoimmunity.
purpose of these studies was to examine whether priming with TNFα and IL-3 Methods: EAU was induced in C57BL/6 (B6) mice by adoptive transfer of
(previously shown to upregulate eosinophil TSLP receptor expression) or co- interphotoreceptor retinoid-binding protein (IRBP)1-20 peptide-specific T cells.
incubation with primary human conjunctival epithelial cells could affect the HMGB1 was then serially identified in the retina by immunohistology and in the
responsiveness of eosinophils to TSLP. aqueous humor (AqH) by ELISA. Mice receiving IRBP-specific T cells were
Methods: Eosinophils were stimulated with various concentrations of TSLP and treated with HMGB1 antagonists and intraocular inflammation documented by
evaluated for release of eosinophil derived neurotoxin by ELISA. For priming funduscopy and histology. Proliferation, cytokine production and the disease
experiments, degranulation was evaluated in the presence and absence of pre- inducing ability of antigen-responder T cells from treated or non-treated mice were
treatment with TNFα and IL-3. For co-incubation experiments, primary human compared.
conjunctival epithelial cells were cultured on transwell filters in 24 well plates. Results: After adoptive transfer of IRBP-specific T cells, intracellular HMGB1 in
Eosinophils were added either apically or basally (and to epithelial cell free, control the inner ganglion cell layer was dramatically reduced within 24h and almost
wells) and challenged with TSLP. completely undetectable in the retina by day 7. By contrast, HMGB1 was
Results: Eosinophils pre-treated with TNFα and IL-3 had enhanced sensitivity to significantly increased in the AqH compared to the control eye. Ocular
TSLP-mediated degranulation with approximately 80-fold less TSLP concentration inflammation induced by IRBP-specific T cells was ameliorated by treatment with
needed to achieve comparable degranulation. Co-incubation of eosinophils with ethyl pyruvate (EP), an antioxidant that suppressesHMGB1 release, or a
conjunctival epithelial cells reduced TSLP-mediated degranulation in both apical neutralizing antibody to HMGB1. The level of HMGB1 in the AqH of treated mice
and basal compartments, suggesting that the response was not contact dependent. was dramatically reduced compared to untreated mice, although proliferation of
Conclusions: Eosinophils exhibit enhanced responsiveness to TSLP when exposed uveitogenic T cells was the same in both mice.
to cytokines present in allergic inflammation. Conversely, epithelial cells may Conclusions: The pathogenicity of autoreactive T cells is dependent on the rapid
provide a stabilizing influence on TSLP-mediated eosinophil degranulation. release of HMGB1 from viable retinal tissue cells. Inhibition of HMGB1 will
Commercial Relationships: Ellen B. Cook, None; James L. Stahl, suppress the development of adoptively transferred EAU.
None; Elizabeth D. Schwantes, None; Sameer A. Mathur, None; Neal P. Commercial Relationships: Guomin Jiang, None; Henry J. Kaplan,
Barney, None None; Huan Yang, None; Deming Sun, None; Amir Reza Hajrasouliha,
Support: NIH Program Project Grant HL088584 None; Hui Shao, None
Support: NIH grants NEI EY12974 (HS), Research to Prevent Blindness (RPB)
Program Number: 1246 Poster Board Number: D1075 Lew R. Wasserman Merit Award (HS), the Department's RPB Unrestricted Grant
Presentation Time: 3:15 PM - 5:00 PM (HK) and the KY Challenge Research Trust Fund (HK).
Inflammatory Cytokines In Thrombospondin-1 Deficient Conjunctiva Block
Goblet Cell Mucin Secretion Program Number: 1248 Poster Board Number: D1077
Laura Contreras-Ruiz, Arpita Gosh-Mitra, Bruce Turpie, Marie Shatos, Darlene A. Presentation Time: 3:15 PM - 5:00 PM
Dartt, Sharmila Masli. Schepens Eye Research Inst and Massachusetts Eye and Ethnic Variation in Uveitis Presentation in Singapore
Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA. Jay J. Siak1,2, Aliza Jap2, Soon-Phaik Chee1A,3. AOcular Inflammation and
Purpose: Ocular surface inflammation in thrombospondin-1 (TSP-1) deficient Immunology, 1Singapore National Eye Centre, Singapore, Singapore; 2Division of
mice is associated with autoimmune Sjögren‟s syndrome, which progressively Ophthalmology, Changi General Hospital, Singapore, Singapore; 3Department of
develops with age. Expression of pro-inflammatory cytokines is detectable in the Ophthalmology, Yong Loo Lin School of Medicine, National University Hospital,
TSP-1null conjunctiva as the mice age. We determined if these cytokines alter Singapore, Singapore.
mucin secretion from mouse conjunctival goblet cells. Purpose: To describe the ethnic variation in uveitis presentation among Chinese,
Methods: The cholinergic agonist pilocarpine was injected intraperitoneally and Malays and Indians in Singapore.
tears collected from WT (C57BL/6) and TSP-1 null mice at 8 and 12 weeks of age. Methods: Cross sectional study using retrospective database and medical chart
Tear MUC5AC content was determined by ELISA. In addition, primary cultures of review of 1236 Singaporeans who consulted the Singapore National Eye Centre
mouse conjunctival goblet cells were grown from WT conjunctival explants for 14 uveitis clinic between 1997 and 2010. Consecutive patients were registered in a
days in complete RPMI medium. Goblet and stratified squamous cell specific uveitis database since 1997. Clinical presentations were described in terms of their

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 by Scientific Section/Group – Immunology/Microbiology (IM)

ocular anatomical involvement, laterality, and known etiology. Prevalence rates Atlanta, GA; 3Ophthalmology, Emory Univ Eye Center, Atlanta, GA;
were age adjusted to year 2010 population census. The likelihood of various 4Ophthalmology, Emory Eye Center, Decatur, GA.
specific etiologies were presented as adjusted odds ratios (OR) adjusting for Purpose: Pediatric uveitis care is challenging and often requires multiple providers
gender, ethnicity and age groups (<21, 21-40, 41-60, >60 years). including pediatric ophthalmology, rheumatology, uveitis, and vitreoretinal
Results: Among all ethnic groups, anterior uveitis (AU) was the commonest, specialists. The study aims to describe baseline visual morbidity, disease
followed by panuveitis, posterior and intermediate uveitis. Among patients with associations, ocular complications, and the management of pediatric uveitis patients
uveitis, anterior uveitis was more common among Chinese (age adjusted 63.7%) from a university-based academic referral center in the Southeast United States.
compared to Malays (47.3%, P=0.003) and Indians (37.5%, P=0.01). AU was more Methods: Retrospective study of patients treated at the Emory Eye Center (EEC)
common among male uveitic patients (67.0% vs 52.5%, P<0.001). Panuveitis was with noninfectious pediatric uveitis from September 2010-present. Demographic
also more common among Malays and Indians with uveitis compared to Chinese information, anatomic classification, systemic diseases, time to referral, baseline
(29.5% vs 14.4%, P=0.003; 26.1% vs 14.4%, P=0.02 respectively). The commonest visual acuity (VA), ocular inflammation, secondary complications, prior
anterior uveitides were HLA B27 AAU, idiopathic AAU and cytomegalovirus treatments, and management strategies following transfer of care were reviewed.
(CMV) AU. Intermediate uveitis were mostly idiopathic and more common among Results: Data from 72 eyes (44 patients) were reviewed from the EEC. The mean
younger age groups (P trend <0.001) and female patients (17.3% vs 7.0%, time to referral 66.5 weeks (Range 1-312 weeks). The mean age was 7.9 years
P<0.001). The commonest posterior uveitides were CMV retinitis, toxoplasmosis, (Range 2-17 years). 67% of patients had bilateral disease and 61% of patients were
tuberculosis (TB)-related uveitis. female. Systemic disease associations were identified in 71% of patients.
The commonest panuveitis uveitides were Vogt-Koyanagi Harada disease, TB and Presenting VA was 20/40 or poorer in 46% of patients. Anterior uveitis was
Behcet‟s disease. Chinese with AU were more likely to have HLA B27 AAU than identified most frequently (50% of eyes), followed by intermediate (35%),
Malays (OR 3.03, 95%CI 1.58-5.79) and Indians (OR 2.34, 95%CI 1.14-4.80). posterior (3%) and panuveitis (13%). Secondary complications were frequent and
They were also more likely to have CMV AU than Malays (OR 3.27, 95%CI 1.18- included posterior synechiae (39%), band keratopathy (15%), and cataracts (29%).
9.07) and Indians (OR 8.64, 95% CI 1.19-62.87). Among patients with panuveitis, Escalation or initiation of systemic immunosuppression was elected as the initial
Indians and Malays were more likely to have TB-related uveitis compared to management decision in 82% of patients. Immunosuppressive medications used
Chinese (OR 7.79, 95%CI 2.36-25.8; OR 7.79, 95%CI 2.74-22.14 respectively). included methotrexate (73%), infliximab (11%), adalimumab (5%) and
Conclusions: There are differences in the commonest uveitides encountered among mycophenolate mofetil (2%). Periocular corticosteroids were elected as the initial
different ethnic groups living in Singapore. therapeutic strategy in a minority of cases (11%).
Commercial Relationships: Jay J. Siak, None; Aliza Jap, None; Soon-Phaik Conclusions: Moderate to severe visual impairment was observed in almost 50%
Chee, None of eyes at referral. Secondary complications were common, potentially reflecting
Support: None significant duration of time prior to referral. Systemic immunosuppression was
initiated or escalated at the initial uveitis appointment in the majority of patients.
Program Number: 1249 Poster Board Number: D1078 Longitudinal follow-up of outcomes will be needed to determine whether subtypes
Presentation Time: 3:15 PM - 5:00 PM of pediatric uveitis respond better to local compared to systemic therapies.
Ethnic Disparities in Uveitis in the Southeastern United States Commercial Relationships: Vincent Y. Ho, None; Sheila Angeles-Han,
Russell W. Read, Kinley Beck, Carrie Huisingh, Gerald McGwin, Jr.. None; G. B. Hubbard, III, None; Steven Yeh, None
Ophthalmology, University of Alabama at Birmingham, Birmingham, AL. Support: Unrestricted grant from Research to Prevent Blindness
Purpose: To determine the role of ethnicity in the epidemiology of uveitis patients
in the Southeastern United States. Program Number: 1251 Poster Board Number: D1080
Methods: The current study was a retrospective chart review of all patients seen Presentation Time: 3:15 PM - 5:00 PM
between 2007 and 2010 (inclusive) at a single tertiary uveitis center in Recurrent Unilateral Crystal-like Deposits Of Unknown Etiology In The
Birmingham, Alabama. Charts were reviewed for demographics, diagnosis, work Anterior Chamber
up, treatment, clinical parameters, and development of complications at each visit. Jennifer H. Hung, Anu P. Gupta, Amilia Schrier, Richard Braunstein. Department
Characteristics were compared between racial subgroups using a t-test and chi- of Ophthalmology, Columbia University, New York, NY.
square test for continuous and categorical variables, respectively. Purpose: To describe a case of unilateral recurrent crystal-like deposits in the
Results: Seven hundred eighty two (782) patients were identified as having uveitis anterior chamber possibly related to medical treatment for rheumatoid arthritis
from the chart review. Of these, 324 (41%) were African-Americans and 434 (56%) Methods: A 57-year old female with no prior ocular history presented with acute
were Caucasians, with 24 (3%) categorized as “other.” A higher proportion of unilateral crystal-like deposits in the anterior chamber of her left eye. Her medical
African Americans were female compared to Caucasians (76% vs. 60%, p<0.0001). history included rheumatoid arthritis, osteoporosis, alcohol abuse and iron-
In terms of anatomical disease distribution, the proportion of posterior cases was 3- deficiency anemia. At initial presentation, the patient was being treated with
fold higher among Caucasians than African Americans (16% vs. 5%, p<0.0001) etanercept. Multiple biopsies of the deposits were performed due to recurrence.
and the proportion of panuveitic cases was almost 3-fold higher among African Results: Electron microscopy, x-ray crystallography, electron-diffraction
Americans than Caucasians (20% vs. 7%, p<0.0001). The associated conditions spectroscopy, and elemental analysis revealed the deposits to be multilayered
more prevalent among Caucasians than African American include HLA-B27 (14% hexagonal bipyramids composed of elements including iron, magnesium,
vs. 6%, p=0.0003), herpetic disease (6% vs. 1%, p=0.0005) and birdshot (4% vs. aluminum and silicon. Progressive corneal decompensation and elevated
0.3%, p=0.0012). However, sarcoid (11% vs. 3%, p<0.0001), persistent post- intraocular pressure necessitated the need for pars plana vitrectomy, valve
operative inflammation (6% vs. 3%, p=0.03), Vogt-Koyanagi-Harada disease (2% placement, repeat deposit excision, cataract extraction and penetrating keratoplasty.
vs. 0.5%, p=0.03) and systemic lupus erythematosus (SLE) (2% vs. 0.5%, p=0.04) Deposit re-accumulation decreased after the discontinuation of etanercept. Further
were more prevalent among African Americans. analysis is pending.
Conclusions: The epidemiology of uveitis varies between ethnic groups. Among Conclusions: This appears to be the first reported case of crystal-like deposits in
African Americans with uveitis, females are more likely to be affected than are the anterior chamber that may occur secondary to anti-tumor necrosis factor
females of Caucasian ethnicity, though Caucasian females are still more likely than medication for rheumatoid
Caucasian males to have uveitis. African Americans are more likely to have arthritis.
panuveitis while Caucasians are more likely to have posterior uveitis. Etiologies of
disease vary along mostly expected patterns, with HLA-B27 disease and birdshot
more common in Caucasians and sarcoid and SLE more common in African
Americans. Interestingly, herpetic disease (simplex or zoster) was significantly
more prevalent in Caucasians than African Americans.
Commercial Relationships: Russell W. Read, None; Kinley Beck, None; Carrie
Huisingh, None; Gerald McGwin, Jr., None
Support: EyeSight Foundation of Alabama; Research to Prevent Blindness
Program Number: 1250 Poster Board Number: D1079
Presentation Time: 3:15 PM - 5:00 PM
Pediatric Uveitis Disease Characteristics And Management In A University-
based, Academic Practice
Vincent Y. Ho1, Sheila Angeles-Han2, G B. Hubbard, III3, Steven Yeh4.
1Ophthalmology, Emory Eye Center, Atlanta, GA; 2Pediatrics, Emory University,

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 by Scientific Section/Group – Immunology/Microbiology (IM)

Commercial Relationships: Supinda Leeamornsiri, None; Paul A. Gaudio,
None; John J. Huang, None
Support: None
Program Number: 1253 Poster Board Number: D1082
Presentation Time: 3:15 PM - 5:00 PM
Necrotizing Scleritis- A Case Series and Review of Literature
Pooja Bhat1, Kanish Mirchia2, Surendra Basti1, Robert Feder1, Dmitry Pyatetsky1.
1Department of Ophthalmology, Northwestern University, Feinberg School of
Medicine, Chicago, IL; 2Government Medical College & Hospital, Chandigarh,
India.
Purpose: Necrotizing scleritis is a rare and severe form of scleritis that can threaten
both vision and integrity of the eye with limited published information on its
presentation,course, treatment and outcome. The purpose of our study is to identify
patients with necrotizing scleritis diagnosed and treated at the Northwestern
University‟s Ophthalmology Department and compare our experience with that
reported in literature.
Methods: Retrospective case-series. Seven patients with necrotizing scleritis were
identified between 2008-2011. Information regarding the duration of scleritis,
underlying autoimmune conditions, prior ocular surgeries, and systemic
immunomodulatory therapy administered was collected. Ocular complications
including impending or actual perforation and its management were recorded. A
review of literature was performed between 1990 and 2011 and thirty two articles
were identified.
Results: In our series, 6 patients had underlying autoimmune diseases which
included rheumatoid arthritis (n=3), and Wegener‟s granulomatosis (n=3). One
patient had surgically induced necrotizing scleritis (SINS) after pterygium excision
with mitomycin C. The average duration of scleritis prior to the onset of necrosis
was 2.8 yrs. Three patients developed bilateral necrosis and 2 had painless necrosis.
Perforation requiring emergent patch grafting occurred in 2 patients. At the onset of
scleral necrosis, intravenous cyclophosphamide (n=2), rituximab (n=1), infliximab
(n=1) and oral methotrexate (n=3) was used in combination with systemic
corticosteroids in all patients. Maintenance was achieved using methotrexate (n=5),
mycophenolate mofetil (n=1) and prednisone (n=1). Complications from systemic
immunomodulation included pancytopenia, pneumonia, and hepatitis which
resolved on cessation of the offending agent. None of the patients developed
recurrent necrosis during their follow-up. Categories among the published reports
were SINS (n=15), autoimmune (n=13), masquerade (n=3) and infection (n=1).
Pterygium excision, rheumatoid arthritis and squamous cell carcinoma were the
most common factors in the first 3 categories while the infectious agent was

Commercial Relationships: Jennifer H. Hung, None; Anu P. Gupta, acanthamoeba. The largest case-series of 4 patients was described in the SINS
None; Amilia Schrier, None; Richard Braunstein, None group while the most common medication used in the autoimmune group was
Support: None infliximab.
Conclusions: Necrotizing scleritis may be associated with life-threatening systemic
conditions requiring potent immunomodulatory therapy and can occur after
Program Number: 1252 Poster Board Number: D1081 uncomplicated ocular surgery. All our patients were successfully managed with a
Presentation Time: 3:15 PM - 5:00 PM systematic approach, newer biologic agents and patch grafting when necessary,
Incidence Of Cystoid Macular Edema After Phacoemulsification With Pars with preservation of the eyeball in all cases.
Plana Vitrectomy In Patients With Uveitis Commercial Relationships: Pooja Bhat, None; Kanish Mirchia,
Supinda Leeamornsiri1, Paul A. Gaudio1,2, John J. Huang1. 1Department of None; Surendra Basti, None; Robert Feder, None; Dmitry Pyatetsky, None
Ophthalmology, Yale Medical School, New Haven, CT; 2Eye Disease Consultants, Support: None
Hartford, CT.
Purpose: To determine the incidence of cystoid macular edema (CME) after
combined phacoemulsification and pars plana vitrectomy in patients with uveitis. Program Number: 1254 Poster Board Number: D1083
Methods: The medical records were reviewed of patients with noninfectious Presentation Time: 3:15 PM - 5:00 PM
uveitis who underwent combined phacoemulsification/pars plana vitrectomy The Amniotic Membrane in Acute Stage IV Ocular Graft versus Host Disease;
between August, 2007 - July, 2011. Patients with diabetes or documented macular Case Report and Review of the Literature
edema at the time of surgery were excluded. Cases were classified according to the Sarah N. Mirza1, Ashley Kingham2A, Amin Alousi2B, Stella K. Kim2A.
presence of postoperative CME within 3 months after surgery. 1Ophthalmology, UT Medical Branch, Galveston, TX; AOphthalmology
Results: 88 surgeries were enrolled. The incidence of postoperative CME was Section/Head and Neck Surgery, BStem Cell Transplantation, 2UT MD Anderson
13.6%. 12 eyes developed CME. Among patients who developed CME, 9 eyes Cancer Center, Houston, TX.
(75%) had anterior uveitis, and 3 eyes (25%) had panuveitis. In non-CME group, Purpose: To describe the first case in the literature of amniotic membrane tissue to
24 eyes (31.6%) had anterior uveitis, 7 eyes (9.2%) had intermediate uveitis, 5 eyes treat acute stage IV ocular graft-versus-host (GVHD) disease.
(6.6%) had posterior uveitis, 40 eyes (52.6%) had panuveitis. In the CME group, 4 Methods: Case report and a literature search.
eyes (33.3%) had history of previous CME, compared with 17 eyes (22.4%) in the Results: A 61 year-old male treated for chronic myelomonocytic leukemia with
non-CME group. In the CME group, 3 eyes (25%) had documented active matched unrelated donor stem cell transplant with severe stage IV skin and GI
inflammation within the 6-month period before surgery, compared with 24 eyes GVHD and on post-transplant day 43 was noted to have extensive bilateral corneal
(31.6%) in the non-CME group. In the CME group, 9 eyes (75%) were treated with epithelial sloughing and dense conjunctival pseudomembranes. Viral cultures were
preoperative corticosteroids prior to surgery, compared with 68 eyes (89.5%) in the negative. Patient was being treated as an inpatient for steroid refractive systemic
non-CME group. Four (33.3%) of the eyes that developed CME underwent GVHD, requiring multiple immunomodulators and photopheresis. The corneal
procedure without intraocular lens implantation, while among patients who did not epithelial defects were slow to heal despite pseudomembrane debridement,
aggressive lubrication and topical steroid therapy. Amniotic membrane in the form
develop CME this figure was 10 (13.2%) eyes. By 3 months, 66.7% of patients in
the CME group achieved visual acuity of 20/40 or better, while in the non-CME of Prokera® amniotic membrane device was inserted into the left eye at the
group this figure was 82.9%. bedside, which had a slightly larger defect than the right eye. During the first 24
Conclusions: In this study of patients with uveitis, the overall incidence of CME hours after insertion of Prokera® amniotic membrane tissue device into the left
after combined phacomulsification/pars plana vitrectomy was 13.6%. eye, the circular corneal epithelial defect showed approximately 50 % reduction in
the left eye as compared to approximately 10 % improvement in the right eye,

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 by Scientific Section/Group – Immunology/Microbiology (IM)

which was treated with conjunctival pseudomembrane debridement, aggressive The RPE cell and its TLRs are strategically situated to provide a rapid innate
lubrication and topical steroids alone. The conjunctival injection also appeared immune defense system for the retina. The RPE cell not only produces pro-
improved in the left eye with the amniotic membrane compared to the right eye inflammatory cytokines important for immune reactivity but also
which received the same dose of topical medication without the amniotic immunosuppressive cytokines that serve to down-regulate immunity, a vital
membrane. 1 day later, Prokera® amniotic membrane device was place in the right mechanism for protecting retinal tissue from excessive damage.
eye. The corneal epithelial defect healed completely in 5 days in the right eye. The Commercial Relationships: Barbara Detrick, None
left eye continued to form extensive pseudomembrane/ membrane, suggesting the Support: RO1DA012777 -NIH/NIDA; RO1 DA 25524 -NIH, NIDA; RO1
presence of worse ocular GVHD in the left eye. With continued debridement, AIO89878-01A1 -NIH/NIAID/NICIH
topical steroid, and Prokera®, the corneal epithelial defect also healed in 8 days in
the left eye. The patient had improved vision during his hospitalization, but
Program Number: 1700
unfortunately did not survive Grade IV systemic GVHD and died in 2 months.
Presentation Time: 2:10 PM - 2:30 PM
There is paucity of published data regarding intervention of acute stage IV ocular
RPE and Immune Privilege
GVHD.
Joan Stein-Streilein. Ophthalmology, Schepens Eye Research
Conclusions: This is the first time the use of amniotic membrane tissue suspended
Institute/Massachusetts Eye and Ear, Boston, MA.
in a forniceal ring (Prokera®) to promote corneal epithelial healing in acute stage
Pigmented epithelial cells of the eye secrete immunosuppressive compounds as
IV ocular GVHD has been reported. In this case, the use of Prokera® was
well as modulate immune cell behavior. Retinal epithelial cells express immune
particularly beneficial in that the device was easily placed at the bedside in an
modulating surface molecules and in vivo are able to modulate activated immune
immunosuppressed patient whose systemic illness precluded a trip to the operating
cells toward T regulatory cells. Immunosuppressive mechanisms of RPE eye cups
room. Prokera® amniotic membrane tissue device can be used to enhance corneal
from healthy mice or mice post retinal laser burn on activated CD4+ CD8+ T cells
epithelial healing in acute stage IV ocular GVHD. A larger series is needed for
are analyzed.
further studies.
Commercial Relationships: Joan Stein-Streilein, None
Commercial Relationships: Sarah N. Mirza, None; Ashley Kingham,
Support: NIH/1R21 EY020614;NIH/R01 EY 11983; DOD:W81XWH-04-1-
None; Amin Alousi, None; Stella K. Kim, None
0892;NIH/R01 EY016476
Support: None
Program Number: 1701
Program Number: 1255 Poster Board Number: D1084
Presentation Time: 2:30 PM - 2:50 PM
Presentation Time: 3:15 PM - 5:00 PM
Immunosuppressive Properties of Retinal Pigment Epithelial (RPE)-induced
Clinical Features and Treatment Outcome of Japanese Patients with Scleritis
Regulatory T cells
Kazuhisa Takahashi, Kuniko Wakayama, Hiroshi Takahashi, Junko Hori.
Manabu Mochizuki. Ophthalmology & Visual Science, Tokyo Med Dental Univ
Ophthalmology, Nippon Medical School, Tokyo, Japan.
Grad School, Tokyo, Japan.
Purpose: To analyze clinical features, systemic associations, treatment in patients
Co-Author: Sunao Sugita, Dept of Ophthalmology & Visual Science, Tokyo
with scleritis.
Medical and Dental University, Graduate School of Medicine, Tokyo, Japan
Methods: Clinical records of 99 patients with scleritis who presented between
Purpose: Retinal pigment epithelium (RPE) cells are able to convert CD4+ T cells
April 2004 and September 2011 to the Ophthalmology at Nippon Medical School,
into T regulatory cells (Tregs) that greatly suppress inflammatory effector cells. In
Tokyo, were reviewed retrospectively.
this study we investigated whether RPE cells and the RPE-induced Tregs have
Results: Of the 99 patients, 57.6% had diffuse anterior scleritis, 20% had
immunosuppressive properties.
episcleritis, 16% had nodular anterior scleritis, 4% had necrotizing anterior
Method: Primary cultured RPE cells were established from normal C57BL/6 mice.
scleritis, and 2% had posterior scleritis. Mean age at presentation was 53.5±15.2
CD4+ T cells were co-cultured with RPE, x-irradiated, and used as regulators (RPE-
(range 22-83 years). Secondary ocular complications were observed in 32% of
induced Tregs). The RPE-induced Tregs were used for the in vitro assay and in
patients, including anterior uveitis in 9%, increased intraocular pressure 31%.
vivo experimental uveitis (EAU) models. In human study, peripheral blood
Systemic disease association was seen in 20% patients, including rheumatoid
mononuclear cells from healthy donors or uveitis patients were co-cultured with
arthritis, tuberculosis, relapsing polychondritis, Wegener‟s granulomatosis,
supernatants from TGFβ2-pretreated human RPE cell lines on anti-CD3-coated
seronegative arthritis, and so on.
plate. Cells were then separated with a CD4+CD25+ regulatory T-cell isolation kit,
100% of episcleritis and 87.7% of diffuse anterior scleritis were treated with only
and co-cultured with the supernatants from RPE, anti-CD3, anti-CD28, high-dose
topical application of steroids and/or immunosuppressive drugs. 6.3% of nodular
recombinant IL-2, and TGFβ2. Expression of CD25high, Foxp3, CD152 (CTLA-4),
anterior scleritis, 75% of necrotizing anterior scleritis, and 100% of posterior
and TNFRSF 18 (GITR) on RPE-induced Tregs was analyzed by flow cytometry.
scleritis needed systemic treatments with oral corticosteroids and/or
Production of cytokines by RPE-induced Tregs was measured by ELISA. In
immunosuppressive drugs. 15.2% of scleritis without systemic disease were treated
addition, proliferation of target Th1/Th17 cells was assessed by [3H]-thymidine
with oral corticosteroids and/or immunosuppressive drugs, biologic agent (anti-
incorporation or CFSE incorporation. Using flow cytometry sorting, these Tregs
TNF agent). On the other hand, 30% of scleritis associated with systemic disease
were separated in order to collect only active Tregs
needed systemic medication.
(CD4+CD25highFoxp3highCD45RA-).
Conclusions: About 90% of diffuse anterior scleritis and nodular anterior scleritis
Results: RPE cells in the presence of TGFβ produce anti-inflammatory effects, and
were treated with only topical application of steroids and/or immunosuppressive
are able to induce Tregs in vitro. Administration of mouse RPE-induced Tregs that
drugs. On the other hand, most of necrotizing anterior scleritis and posterior
greatly expressed Foxp3 significantly suppressed ocular inflammation in EAU
scleritis received oral corticosteroids and/or immunosuppressive drugs, biologic
models. The RPE-induced Tregs were able to suppress retinal antigen-specific T
agent. Our report suggested that scleritis associated with a systemic disease needs
cells (Th1/Th17 response) in vitro. As well as murine Tregs, human RPE-induced
oral corticosteroids and/or immunosuppressive drugs, biologic agent (anti-TNF)
Tregs expressed higher levels of the Treg cell markers CD25high, Foxp3, CD152,
more often than scleritis without systemic disease.
and TNFRSF 18, and RPE-induced Tregs produced TGFβ1 and IL-10, but not
Commercial Relationships: Kazuhisa Takahashi, None; Kuniko Wakayama,
inflammatory cytokines such as IFNγ, IL-17, and TNFα. In addition, human RPE-
None; Hiroshi Takahashi, None; Junko Hori, None
induced Tregs included significant numbers of CD4+CD25highFoxp3highCD45RA-
Support: Grants-in-Aid for Scientific Research (C) from Japan Society for the
active Tregs that significantly suppressed activation of Th1/Th17 cell lines,
Promotion of Science
indicating that they have immunosuppressive properties. Furthermore, we are able
to remove CD4+CD25lowFoxp3lowCD45RA- non-suppressing cytokine-secreting T
248 Retinal Pigment Epithelium Orchestrates Retinal Immunobiology - cells from the in vitro-manipulated Treg population.
Minisymposium Conclusions: RPE cells and the RPE-induced Tregs have immunosuppressive
Monday, May 7, 2012, 1:45 PM - 3:30 PM properties in the eye. Thus, these data suggest that Tregs might be useful as
Floridian A Symposium individualized therapy for ocular inflammatory diseases.
Program #/Board # Range: 1699-1703 Commercial Relationships: Manabu Mochizuki, None
Organizing Section: Immunology/Microbiology Support: Grant-in-Aid for Scientific Reseach No19390440, Japan
Contributing Section(s): Retinal Cell Biology,Retina+
Program Number: 1702
Program Number: 1699 Presentation Time: 2:50 PM - 3:10 PM
Presentation Time: 1:50 PM - 2:10 PM RPE Immunomodulating Neuropeptides
Innate Immunity in the Retina: TLR signaling in RPE Cells Andrew W. Taylor. Ophthalmology, Boston University School of Medicine,
Barbara Detrick. Pathology, The Johns Hopkins University, SOM, Baltimore, MD. Boston, MA.

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 by Scientific Section/Group – Immunology/Microbiology (IM)

The healthy RPE monolayer is a source of soluble immunomodulating Commercial Relationships: Homayon Ghiasi, None; Sariah Allen,
neuropeptides and cytokines that suppress endotoxin-stimulated inflammatory None; Kevin Mott, None; Antje Rhode-Kurnow, None; Xianzhi Jiang,
activity in macrophages. In addition, the RPE monolayer produces two None; Dale Carpenter, None; Clinton Jones, None; Steven WEchsler,
neuropeptides that induce myeloid suppressor cell (MSC)-like activity in resting None; Carl Ware, None
macrophages. In contrast, disrupted RPE cell layer of wounded retinas, or of uveitic Support: NIH Grants EY13615 and AI067890.
eyes do not suppress macrophage inflammatory activity, nor mediate induction of
MSC. In parallel, there is a change in the retina from MSC-like microglia cells into Program Number: 2233
inflammatory microglia cells. This demonstrates that an important part of Presentation Time: 4:00 PM - 4:15 PM
neuroimmunomodulation within the eye is a requirement for a healthy intact RPE Acute Zonal Accult Outer Retinopathy (azoor) Is A Form Of Autoimmune
monolayer. It also implies that changes in RPE health because of trauma, and aging Retinopathy
has immunological consequences that can contribute to other ocular diseases. John R. Heckenlively, Angeline Wang, A. J. Karoukis, Jennifer Melms, Richard
Commercial Relationships: Andrew W. Taylor, None Hackel, Kari Branham, Naheed W. Khan. Ophthal & Vis Sciences, Univ of
Support: The Massachusetts Lions Eye Research Foundation, and NIH Grant Michigan-Kellogg Eye Ctr, Ann Arbor, MI.
EY010752 Purpose: To study 25 cases of AZOOR whose diagnosis was strictly based on
clinical descriptions by J. Donald Gass1, to examine clinical characteristics, visual
Program Number: 1703 field scotomata patterns, and measure presence or absence of anti-retinal
Presentation Time: 3:10 PM - 3:30 PM antibodies.
Transplanted RPE Survival on Aged and AMD Bruch's Membrane Methods: Twenty-five patients with histories of sudden visual loss, photopsias and
Marco Zarbin. Institute of Ophthalmology & Visual Science, UMDNJ New Jersey appearance of missing areas in their vision were assessed for AZOOR based on the
Medical School, Newark, NJ. characteristic onset, abnormal ERGs, and scotomatous changes seen on
Commercial Relationships: Marco Zarbin, Advanced Cell Technology (F), standardized visual field testing. The presence of enlarged blindspots, or large
Alimera (C), Allergan (C), Celgene (C), Eli Lilly (C), Foundation Fighting scotomatous areas contiguous with blindspots were definitive criteria for
Blindness (R), Iridex (C), Novartis (C), Pfizer (C), US 12/738839 (P), US classification of AZOOR. Several patients with large scotomata in peripheral
61/452,458 (P), US 61/452,487 (P), US 61/471,951 (P) retina, as described by Gass, were also designated as cases of AZOOR. Western
Support: Foundation Fighting Blindness blots were used to detect anti-retinal antibodies in a standardized fashion.
Results: The majority of AZOOR patients were women 21/25 (84%) and 15/25
(60%) of the patients had personal or family histories of autoimmune disease.
275 CNS and Retinal Inflammation and Infection
Western blots from AZOOR patients showed an average of 6.5 immuno-reactive
Monday, May 7, 2012, 3:45 PM - 5:30 PM
bands compared to 4.5 for a comparative group of 25 autoimmune retinopathy
Room 315 Paper Session
patients. Characteristic visual field changes included enlarged blind spots (15/50
Program #/Board # Range: 2232-2238
eyes), central scotomata, and large scotomata connecting to or contiguous to the
Organizing Section: Immunology/Microbiology
blindspot (24/34), and visual field isopter contraction (18/34) was common. We
Contributing Section(s): Retina+,Retinal Cell Biology
examined the retinal changes associated with the enlarged blindspots, reviewing
fluorescein angiograms or autofluorescent photographs in corresponding areas; we
Program Number: 2232 found peripapillary retinal changes (staining or depigmentation) often matching the
Presentation Time: 3:45 PM - 4:00 PM shape of the scotomata found on Goldmann visual field, suggesting that anti-retinal
Interplay of LAT and HVEM affect HSV-1 Latency and CD8+ T Cell antibodies are accessing the retina and subretinal space via the optic canal where
Exhaustion there is frequently a loss in the RPE confluence and their tight junctions normally
Homayon Ghiasi1, Sariah Allen1, Kevin Mott1, Antje Rhode-Kurnow2, Xianzhi protecting the retina.
Jiang3, Dale Carpenter3, Clinton Jones4, Steven WEchsler3, Carl Ware2. Conclusions: This study strongly suggests that AZOOR is a form of acute-onset
1Surgery/Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los autoimmune retinopathy, in which the distinctive visual field patterns, in part, are
Angeles, CA; 2Infectious and Inflammatory Diseases Center, Laboratory of the result of a breakdown of the blood-retinal barrier at the optic canal or
Molecular Immunology, Sanford|Burnham Medical Research Institute, La Jolla, peripapillary retina, allowing spread of anti-retinal antibodies in the subretinal
CA; 3Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA; space where they can cause damage to the RPE and photoreceptors directly,
4Department of Veterinary and Biomedical Sciences, University of Nebraska, depending on their antigenic targets. Funded investigations into all forms of
Lincoln, NE. autoimmune retinopathy are badly needed, since knowledge-based treatments are
Purpose: The herpesvirus entry mediator (HVEM; TNFRSF14) plays a significant likely save vision in these patients. 1 Am J Ophthalmol. 2002 Sep;134(3):329-39
role regulating immune responses, yet appears to have only a minor role as an viral Commercial Relationships: John R. Heckenlively, None; Angeline Wang,
entry route during primary infection in mucosal and neural tissues. The immune None; A. J. Karoukis, None; Jennifer Melms, None; Richard Hackel,
regulatory role for HVEM suggested the possibility of its involvement in latency. None; Kari Branham, None; Naheed W. Khan, None
Latency associated transcript (LAT) of HSV-1 plays a major role in the level of Support: Foundation Fighting Blindness, Research to Prevent Blindness
HSV-1 latency and virus reactivation. Whether there is a synergy between LAT and
HVEM in the establishment of latency and reactivation in trigeminal ganglia (TG)
Program Number: 2234
of infected mice is unknown. To address this issue, we used both an in vitro model
Presentation Time: 4:15 PM - 4:30 PM
looking at the effect of LAT on HVEM in Neuro2A and C1300 cells and LAT(+)
Influence Of The Intestinal Microbiota In The Development Of Spontaneous
and LAT(-) viruses to evaluate the effect of LAT in HVEM-deficient mice.
Autoimmune Uveitis
Methods: WT (C57BL/6) and HVEM-/- mice were infected ocularly with 2 X 105 Carlos R. Zarate-Blades1A, Reikp Horai1A, Jun Chen1A, Phyllis B. Silver1A, Chi-
PFU/eye of WT (LAT-plus) HSV-1 strain McKrae or dLAT2903, dLAT-CD80 and Chao Chan1B, Rachel R. Caspi1A. ALaboratory of Immunology, BImmunopathology
dLATgK3 (LAT-minus viruses). Individual TG from surviving mice were isolated Section, 1National Eye Institute, NEI-NIH, Bethesda, MD.
on day 30 post-infection (PI) and used for detection of viral DNA. Isolated TG
Purpose: Involvement of microorganisms in triggering human autoimmune uveitis
were also tested for the presence of viral antigens and exhaustion markers by
has been repeatedly suggested based on anecdotal evidence, but has never been
FACS, RT-PCR, and immunostaining. In addition, the effect of LAT on up-
proven. Recently, we developed an IRBP-specific T cell receptor (TCR) transgenic
regulation of HVEM in Neuro2A cells stably expressing or transiently transfected
mouse (R161H) that develops autoimmune uveitis spontaneously by 2 months of
with various LAT regions were evaluated.
age. We previously reported that R161H mice treated with broad-spectrum
Results: The increase of latency in TG of WT mice infected with LAT(+) virus
antibiotic to reduce intestinal flora experienced a concomitant reduction of retinal
compared to LAT(-) virus correlated with an increase in the level of HVEM, but
inflammation. The aim of this project is to understand how the microbiota regulate
not nectin-1, nectin-2, PILR-α, or 3-O sulfated heparin sulfate. LAT also
the immune response and thereby modulate the development of spontaneous uveitis
upregulated HVEM expression in Neuro2A cells stable expressing LAT, and in
Methods: R161H mice were kept untreated or received the antibiotic cocktail
cells transfected with LAT sRNA1 or sRNA2. In contrast to WT mice, the level of
AMNV (ampicillin, metronidazole, neomycin and vancomycin) in the drinking
latency in HVEM-/- mice infected with LAT(+) was three fold lower, similar to that
water continuously throughout life. Uveitis was evaluated by fundus examination.
of LAT(-) virus. Decrease of latency in HVEM-/- mice or WT mice infected with
Bacterial composition in fecal pellets was determined by 16S rRNA gene detection.
LAT(-) viruses correlated with a significant decline in the level of CD8 and PD-1.
Immune cell profiles in lymphoid tissues and intestinal lamina propria were
However, the absence of HVEM did not alter primary virus replication in the eye of
examined by flow cytometry
HVEM-/- mice compare with HVEM+/+.
Results: Analysis of the gut microflora revealed that mice receiving the AMNV
Conclusions: These results demonstrate for the first time a link between LAT and
cocktail had a substantially decreased number of culturable species and a reduced
HVEM suggesting a molecular basis for selective advantage of this entry route for
bacterial mass as defined by the relative amount of 16S rRNA. This was
the pathogen during latency-reactivation.

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].