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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMulticentric study validation of a new molecular method forLimbal Stem Cell Deficiency diagnosis based on MUC5ACtranscript detection in corneal epithelium by reverse dot-blotstripTatiana M. Suarez-Cortes 1 , Iker Garcia 1 , Jaime Etxebarria 2 , JesusMerayo-Lloves 3 , Josep Torras 4 , Ana Boto-de-los-Bueis 5 , David Diaz-Valle 6 , Rosalia Mendez 6 , Xabier Landaluce 1 , Arantxa Acera 1 .1 Biomed R & D, Bioftalmik, Derio, Spain; 2 Department ofOphthalmology, Cruces Hospital, Plaza Cruces 12, Baracaldo, Spain;3 Fundación de Investigación Oftalmológica, Oviedo, Spain;4 Department of Ophthalmology, Hospital Clinic de Barcelona,Barcelona, Spain; 5 Department of Ophthalmology, Hospital La Paz,idiPaz, Madrid, Spain; 6 Department of Ophthalmology, HospitalClínico San Carlos, Madrid, Spain.Purpose: To validate the efficacy, accuracy and sensitivity of a PCRstripbased on reverse dot-blot for detection of MUC5AC transcriptas indicative of the presence of goblet cells in cornea of patients withLimbal Stem Cell Deficiency (LSCD), and to evaluate the correlationwith clinical diagnosis.Methods: Eighty-five corneal impression cytology (IC) samplesfrom 54 subjects were analyzed in the study: 44 clinically diagnosedLSCD corneas and 41 healthy corneas. Sixteen conjunctivalimpression cytology (IC) samples were assayed as positive control. Atotal of 101 impression cytology samples were analyzed in the study.All the corneal impression cytologies were processed by RNAextraction, retrotranscription, and analyzed by the presence of gobletcells in the cornea by the PCR-strip based on reverse blotting system(Limbokit).Results: The total IC samples analyzed indicated that 43 of 44samples clinically diagnosed as LSCD were confirmed positive forMUC5AC, 33 of 41 healthy corneas were confirmed negative forMUC5AC, 4 healthy corneas were found positive, and 4 wererendered inconclusive results. All conjunctival impression cytologiesused as positive control were confirmed MUC5A positive. The dataindicate a global correlation of 91.1%. (p=0.0001). Confirmation ofPCR results in TAE-agarose gels indicated that reverse dot-blot stripwas more sensitive for bands detection and enhances visualization ofresults. The overall sensitivity, specificity, Positive Predictive Value(PPV) and Negative Predictive Value (NPV) of Limbokit visualizedon PCR-strips, were 98%, 89%, 91% and 97% respectively.Conclusions: The PCR-strip test based on reverse blotting was foundto be a sensitive technique for detection of MUC5AC transcript incorneal epithelium. The test results correlate well with clinicaldiagnosis of characterized LSCD cases. The overall sensitivity,specificity, and positive and negative predictive values weresatisfactory for diagnostic purposes. The PCR-strip based test(Limbokit) constitutes a robust system for the detection of MUC5ACin corneal epithelium and may be used for early detection and formild cases of Limbal Stem Cell Deficiency, and also as an objectiveclinical tool for monitoring of treatments and surgical decisions.Commercial Relationships: Tatiana M. Suarez-Cortes, BioftalmikS.L. (E); Iker Garcia, Bioftalmik Applied Research (P); JaimeEtxebarria, bioftalmik (C); Jesus Merayo-Lloves, Ferrara & HijosSL (I); Josep Torras, None; Ana Boto-de-los-Bueis, None; DavidDiaz-Valle, None; Rosalia Mendez, None; Xabier Landaluce,Bioftalmik (E); Arantxa Acera, BIOFTALMIK SL (E)Support: NEOTEC Program, Grant IDI-20080118Program Number: 548 Poster Board Number: B0185Presentation Time: 10:30 AM - 12:15 PMOptimized culturing conditions for limbal epithelial cellscultivated on semi-synthetic collagen matricesCorinna Petsch 1 , Ursula Schlotzer-Schrehardt 1 , Markus Frey 2 ,Johannes Menzel-Severing 1 , Friedrich E. Kruse 1 , Bjoern O.Bachmann 1 . 1 Department of Ophthalmology, University of Erlangen-Nuremberg, Erlangen, Germany; 2 RESORBA WunversorgungGmbH & Co.KG, Nuremberg, Germany.Purpose: To optimize culturing conditions for limbal epithelial cellson variants of a semi-synthetic collagen matrices with different invivo degradation characteristics.Methods: Limbal epithelial stem cells were clonally enriched on 3T3feeder cells and subcultivated on 3 variants (two crosslinked and onenon-crosslinked variant) of a semi-synthetic type I collagen substrate(RESORBA, Germany). For clonal enrichment and subcultivation onthe collagen matrices 3 different cell culture media were evaluated:MCDB151, DMEM/F12-1 (with bovine pituitary gland extract) andDMEM/F12-2 (without bovine pituitary gland extract). After fixationcell cultures were examined concerning cell adhesion, proliferationand cellular phenotype by light and electron microscopy as well asimmunohistochemistry.Results: Immunohistochemistry as well as light and electronmicroscopy revealed no differences in adhesion, proliferation and cellsheet formation between cell cultures on either variant of the collagenmatrix. When cultured with MCDB151 or DMEM/F12-1 monolayerformation of limbal epithelial cells was seen, while the use ofDMEM/F12-2 resulted in a multilayered cell sheet.By immunohistochemistry, E-Cadherin (as a marker for adherensjunctions) and K3/12 (as a differentiation marker) were localized inall cell layers. Integrinα6 (marker for hemidesomsomes) and p63 (aputative stem cell marker) were expressed in the basal cell layer. P63was also apparent in upper layers of cells cultured on non-crosslinkedcollagen matrices.Electron microscopically hemidesmosomes were seen on cells of thebasal cell layer of cultures on either collagen substrate when culturedwith DMEM/F12-1 or DMEM/F12-2.Conclusions: Crosslinked and non-crosslinked variants of a semisyntheticcollagen matrix are suitable for the cultivation of limbalepithelial cells. The use of DMEM/F12-2 with either collagen matrixresulted in a multilayered cell sheet of cultured limbal epithelial cells.The ability to serve as a growth substrate for limbal epithelial cellsand its known biocompatibility with the cornea indicates that the usedcollagen matrices might be suitable for cultivation and transplantationof ex vivo expanded limbal epithelial cells.Commercial Relationships: Corinna Petsch, RESORBAWundversorgung GmbH &Co.KG, Nuremberg, Germany (F); UrsulaSchlotzer-Schrehardt, None; Markus Frey, RESORBA MedicalGmbH (E); Johannes Menzel-Severing, None; Friedrich E. Kruse,None; Bjoern O. Bachmann, NoneSupport: Medical Valley EMR Erlangen Project A-06, sponsored bythe Federal Ministry of Education and Resarch, Germany (BMBF)Program Number: 549 Poster Board Number: B0186Presentation Time: 10:30 AM - 12:15 PMCD45+F4/80+ immune cells are rapidly recruited to the woundedge after corneal debridement woundsGauri Tadvalkar 1 , Sonali Ghosh 1 , Ahdeah Pajoohesh-Ganji 1 , JaniceL. Walker 2 , A S. Menko 2 , Mary Ann Stepp 1 . 1 Anatomy &Regenerative Biology, George Washington University MedicalCenter, Washington, DC; 2 Pathology, Anatomy and Cell Biology,Thomas Jefferson University, Philadelphia, PA.Purpose: A population of leader cells was found to direct migrationof lens epithelial cells in response to wounding. The current studieswere carried out to determine 1) whether leader cells are involved inthe response of the corneal epithelium to injury, and 2) test thehypothesis that leader cells are immune cells.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: 1.5 mm debridement wounds were made manually in 8week old BALB/c using one of two techniques: a rotating burr (RB)or a dulled blade (DB). Mice were either sacrificed at the time ofwounding (Time 0, T0) or allowed to heal for 1 or 6 hrs. All eyeswere fixed within 5-10 min after injury. Corneas were used for wholemount immunofluorescence studies to localize CD45 (the commonleukocyte antigen), F4/80 (a monocyte marker), GL3 (an epitopepresent on γδT cells), and vimentin, at the leading edges of thewounded corneas. For each variable studied, no fewer than 3 corneasfrom 3 different mice were used.Results: CD45+ cells appear at the leading edge in the T0 corneas.T0 DB wounds recruit more F4/80+ cells compared to RB woundsand this difference persists through the 6 hr time point. Similar levelsof F4/80+ cells were present at T0 and 1 hr after wounding. Therewere several detectable vimentin+GL3+ cells present at the leadingedge at T0 and 1 hr post-injury for both RB and DB wounds. Bycontrast, after 6 hrs, GL3+ cells were absent after DB, and only rarelyseen after RB wounding.Conclusions: The rapid migration of leader cells to the wound edgefollowing mock cataract surgery in the lens is also seen in the corneaafter manual debridement wounding. The numbers of leader cellsrecruited at T0 and 6 hrs after wounding varied depending on the typeof wound. All of the leader cells are positive for CD45, with themajority being positive also for F4/80. A subpopulation of the leadercells are vimentin+GL3+ but these cells are more transient than theF4/80+ cells since they are largely absent by 6 hrs after wounding.Unlike the mouse cornea, the majority of the leader cells in thechicken lens wound model express vimentin. While the functions ofthese rapidly appearing immune cells is not clear, our studies indicatethat wound healing in chicken and mouse and in diverse tissuesincluding the lens and cornea share a similar property: rapidrecruitment of immune derived cells to the leading edge.Commercial Relationships: Gauri Tadvalkar, None; SonaliGhosh, None; Ahdeah Pajoohesh-Ganji, None; Janice L. Walker,None; A S. Menko, None; Mary Ann Stepp, NoneSupport: EYO08512 (MAS), EY021784 (ASM,MAS)Program Number: 550 Poster Board Number: B0187Presentation Time: 10:30 AM - 12:15 PMBasement Membrane Removal Reduces Inflammation in theMouse Cornea and Enhances Wound HealingSonali Pal-Ghosh 1 , Ahdeah Pajoohesh-Ganji 1 , Gauri Tadvalkar 1 ,Daniel R. Saban 2 , Mary Ann Stepp 1 . 1 Anatomy & RegenerativeBiology, George Washington University Medical Center,Washington, DC; 2 Ophthalmology and Immunology, DukeUniversity School of Medicine, Durham, NC.Purpose: An in vivo mouse model has been developed thatreproducibly induces recurrent epithelial erosions in wild-type micespontaneously within two weeks after a single 1.5 mm cornealdebridement wound using a dulled blade. Using a rotating burr ratherthan a dulled blade to create a 1.5 mm wound allows mice to healwithout developing erosions. These experiments were conducted todetermine the cause of the difference in healing outcomes after dulledblade compared to rotating burr wounds.Methods: A 1.5 mm area of corneal epithelium was removed fromthe corneal surface of adult C57BL/6 mice using either a dulled bladeor rotating burr. Corneas were allowed to heal in vivo for 0, 3, 6 hr or5 days. After sacrifice, tissues were used for immunofluorescence,QPCR, flow cytometry, and/or chemokine protein array studies. Flowexperiments were repeated three times. QPCR was performed oncontrol (n=8) and 6 hr dulled blade or rotating burr wounded (n=8)corneas. For QPCR studies, RNA isolated from epithelial cells onlyand whole dissected corneas were compared. All experiments wererepeated at least twice.Results: Data show that 1) erosions form after dulled blade but notafter rotating burr wounds, 2) the basement membrane (BMZ) is leftbehind after dulled blade wounds but is removed by the rotating burr,3) there are more monocytes (CD45+/ Ly6C hi/ Ly6G+/ CD11b hi/F480 low/ CD11c+) and γδ T cells (CD45+/GL3 hi) recruited 6 hrafter dulled blade wounds, and 4) chemokine array studies show that,at the time of wounding and 5 days later, chemokines are elevatedafter dulled blade compared to rotating burr wounds.Conclusions: Despite the fact that rotating burr wounds remove theBMZ, damage more of the subbasal nerves, and induce morecytokine mRNAs within corneal epithelial cells, they heal withoutdeveloping erosions. Improved healing after trauma is associatedwith basement membrane removal and the reduced influx ofmonocytes and γδT cells 6 hr after wounding. Data from chemokinearrays indicate more CCL8 and CCL12 and complement proteins (5aand Factor D) retained on the corneal stroma after dulled bladewounds that may account for the early increase in monocyte and γδTcells seen after dulled blade wounds.Commercial Relationships: Sonali Pal-Ghosh, None; AhdeahPajoohesh-Ganji, None; Gauri Tadvalkar, None; Daniel R.Saban, Schepens Eye Res Inst, Mass Eye and Ear, (P), ElevenBiotherapuetics (R); Mary Ann Stepp, NoneSupport: EYO08512 (MAS), EY021784 (ASM,MAS)Program Number: 551 Poster Board Number: B0188Presentation Time: 10:30 AM - 12:15 PMSchirmer Testing and Corneal Surface Findings in ChronicGraft-Versus-Host DiseaseKathleen M. Williamson 1 , Evan Allan 3 , Michelle P. Lin 2 , Michael C.Wu 1 . 1 Ophthalmology, University of Washington, Seattle, WA;2 Ophthalmology, Cleveland Clinic, Cleveland, OH; 3 Ophthalmology,University of Oklahoma, Oklahoma City, OK.Purpose: To report whether low Schirmer testing scores arepredictive of ocular surface findings in patients with chronic graftversus-hostdisease (cGVHD) diagnosed after hematopoietic celltransplantation.Methods: Retrospective study of 429 consecutive patients withcGVHD referred for ophthalmic examination between 1990 and2006. Office visit records were reviewed for extraction of dataincluding visual acuity, Schirmer testing scores, slit-lampbiomicroscopic findings (including corneal staining with fluorescein),and fundoscopic abnormalities. In this analysis, we considered therelationship between Schirmer testing scores and corneal staining.Two arbitrary cutoff values were used to define ‘low’ Schirmerscores, 5mm or less and 10mm or less. Analysis was then performedto determine if there was an association between ‘low’ scores andcorneal surface staining.Results: In our population of patients with cGVHD, Schirmer testingscores of 5 mm or less are associated with presence of cornealfluorescein staining (p
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