30th Biennial Cornea Conference Program

30t h B ien n ia l

Cornea Conference

October 12-14, 2017 OCTOBER 12-14, 2017Boston, MA

Boston, MA

Funding for this conference was made possible in part by the National Eye Institute (1 R13 EY028423-01).

The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention by trade names, commercial practices, or organizations imply endorsement by the U.S. government

3 0 th B i e n n i a l

Cornea Conference Featuring a Celebration of Claes H. Dohlman, MD, PhD

October 12-14, 2017 Boston

AFFILIATES: Massachusetts Eye and Ear • Schepens Eye Research Institute of Mass. Eye and Ear • Massachusetts General Hospital Boston Children’s Hospital • Beetham Eye Institute at the Joslin Diabetes Center • Brigham and Women’s Hospital Beth Israel Deaconess Medical Center • Cambridge Health Alliance • VA Boston Healthcare System • VA Maine Healthcare System PARTNERS: Aravind Eye Hospital (India) • LV Prasad Eye Institute (India) • Shanghai Eye and ENT Hospital, an affiliate of Fudan University (China)

eye.hms.harvard.edu

Table of Contents 4 Organizing Committees 5 Welcome Letter 7 Program Agenda 15 Participant Biographies and Abstracts 61 Poster Abstracts

Harvard Department of Ophthalmology

Organizing Committees Co-Chairs Reza Dana, MD, MSc, MPH Harvard Ophthalmology/Mass. Eye and Ear Ula Jurkunas, MD Harvard Ophthalmology/Mass. Eye and Ear Research Conference Committee (Friday, October 13) Pablo ArgĂźeso, PhD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear Sunil Chauhan, DVM, PhD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear Darlene Dartt, PhD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear

Jules Baum, MD Tufts-New England Medical Center, Emeritus Jessica Ciralsky, MD Weill Cornell Medicine Kathryn Colby, MD, PhD University of Chicago Medical Center Ilene Gipson, PhD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear

Claes H. Dohlman, MD, PhD Harvard Ophthalmology/Mass. Eye and Ear Emeritus

Peter Laibson, MD Wills Eye Hospital, Emeritus

Ilene Gipson, PhD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear

Deborah Langston, MD Harvard Ophthalmology/Mass. Eye and Ear Emerita

Deborah Jacobs, MD Boston Foundation for Sight and Harvard Ophthalmology/Mass. Eye and Ear Ahmad Kheirkhah, MD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear Noorjahan Panjwani, PhD Tufts University School of Medicine Eleftherios Paschalis, PhD Harvard Ophthalmology/Schepens Eye Research Institute of Mass. Eye and Ear Andrew Taylor, PhD Boston University School of Medicine Vickery Trinkaus-Randall, PhD Boston University School of Medicine

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A Tribute to Claes H. Dohlman Committee (Saturday, October 14)

Administrative Team Harvard Ophthalmology Cornea Center of Excellence Bonnie Brodowski, MBA Erica Eaton, MLS Leila Foster, MPH

30th Biennial Cornea Conference Welcome to the 30th Biennial

Cornea Conference

S

ince the 1960s, the Biennial Cornea Conference has been the premier global cornea and ocular surface academic research conference. Today, more than 200 leaders and trainees from academia and industry have come together to explore current basic, clinical, and translational research developments. The event facilitates open dialogue, collaboration, and scientific inquiry, with the ultimate goal of accelerating cures for blinding corneal diseases.

This year, the conference will focus on ocular surface, immunology and microbiology, endothelial cell biology, and innovation and new techniques.

Additionally, to commemorate the 30th year of the conference, the Saturday program is dedicated to Dr. Claes H. Dohlman—one of the most influential leaders in cornea research, clinical care, and education. In a career that spans nearly six decades, Dr. Dohlman is internationally recognized as the founder of modern corneal science. The program will feature presentations from past Dohlman fellows and the Claes H. Dohlman Lecturer.

Thank you in advance for your active participation, which will greatly contribute to the success of this forum and will help us identify novel avenues for future exploration. We look forward to some stimulating discussions!

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30th Biennial Cornea Conference

Program Agenda

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Harvard Department of Ophthalmology

Poster Session Thursday, October 12, 2017 5:30-8:30pm

Scientific Poster Session and Reception Hiebert Lounge Boston University School of Medicine

Scientific Program Friday, October 13, 2017 7:15 – 7:45am

Breakfast and Registration

7:45 – 8:00am

Welcome and Introduction

Reza Dana, MD, MSc, MPH Claes H. Dohlman Professor of Ophthalmology Committee on Immunology, Harvard Medical School Director, Cornea and Refractive Surgery Service, Mass. Eye and Ear Senior Scientist, Schepens Eye Research Institute of Mass. Eye and Ear

8:00 – 9:40AM

SESSION 1: OCULAR SURFACE

Moderated by Pablo Argüeso, PhD

8:00 – 8:20am Galectin-8 Modulates Corneal Lymphangiogenesis and Infection Noorjahan Panjwani, PhD Professor of Ophthalmology and Developmental, Molecular, and Chemical Biology Director of Research, Department of Ophthalmology, Tufts University School of Medicine 8:20 – 8:40am

ecorin+PEDF Gene Therapy Eliminates Corneal Neovascularization D and Restores Vision in vivo Rajiv R. Mohan, MSc, PhD Professor of Ophthalmology and Molecular Medicine University of Missouri, Columbia

8:40 – 9:00am

Sex, Pain, & Microglia Michael Salter, MD, PhD, FRSC Senior Scientist and Chief of Research The Hospital for Sick Children Senior Scientist in the Program in Neurosciences & Mental Health Professor of Physiology University of Toronto

9:00 – 9:20am

he Corneal Epithelium and its Role in Maintaining the Sensory Nerves T Mary Ann Stepp, PhD Professor of Anatomy and Regenerative Biology George Washington School of Medicine and Health Sciences

9:20 – 9:40am

Panel Discussion

9:40 – 10:00AM

BREAK

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30th Biennial Cornea Conference

10:00AM – 12:00PM

SESSION 2: ENDOTHELIAL CELL BIOLOGY

Moderated by Ula Jurkunas, MD

10:00 – 10:20am

ridging the Gap in the Etiology of Fuchs’ Endothelial Corneal Dystrophy B Shivakumar Vasanth, PhD Instructor in Ophthalmology, Harvard Medical School Investigator, Schepens Eye Research Institute of Mass. Eye and Ear

10:20 – 10:40am

ndothelial Dystrophy in the Slc4a11 Knock-Out Mouse E Joseph Bonanno, OD, PhD Professor and Dean Indiana University School of Optometry

10:40 – 11:00am

Future Realities in Corneal Endothelial Regenerative Medicine Shigeru Kinoshita, MD, PhD Professor and Chair Department of Frontier Medical Science and Technology for Ophthalmology Kyoto Prefectural University of Medicine

11:00 – 11:20am Elucidating the Genetic Basis of Posterior Polymorphous Corneal Anthony Aldave, MD Professor of Ophthalmology, Walton Li Chair in Cornea and Uveitis Chief, Cornea and Uveitis Division Director, Cornea and Refractive Surgery Fellowship UCLA Stein Eye Institute 11:20 – 11:40am

uchs’ Dystrophy and Corneal Endothelium from Genetics to Stem Cells F and Regeneration Natalie Afshari, MD, FACS Professor of Ophthalmology Stuart I. Brown, MD, Chair in Ophthalmology in Memory of Donald P. Shiley Chief, Division of Cornea and Refractive Surgery Vice Chair of Education, Shiley Eye Institute at UC San Diego Health

11:40am – 12:00pm

Panel Discussion

12:00 – 1:15PM

LUNCH

1:15 – 3:00PM

SESSION 3: IMMUNOLOGY AND MICROBIOLOGY

Moderated by Andrew Taylor, PhD

1:20 – 1:40pm Corneal Plasmacytoid Dendritic Cells—The New Cells on the Block Pedram Hamrah, MD Associate Professor of Ophthalmology Director, Center for Translational Ocular Immunology Tufts University School of Medicine 1:40 – 2:00pm Pivotal Role of Dynamin in Adenovirus Trafficking in Corneal Cells Jaya Rajaiya, PhD Assistant Professor of Ophthalmology, Harvard Medical School Assistant Scientist, Mass. Eye and Ear 2:00 – 2:20pm Immunological Graft Rejections After DMEK Claus Cursiefen, MD Chairman and Professor, Department of Ophthalmology University of Cologne, Germany

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Harvard Department of Ophthalmology

2:20 – 2:40pm Neuropeptide Mediated Regulation of the Ocular Surface Homeostasis and Herpes Stromal Keratitis Susmit Suvas, PhD Associate Professor, Departments of Ophthalmology, Anatomy & Cell Biology, and Immunology and Microbiology Wayne State University School of Medicine 2:40 – 3:00pm

Panel Discussion

3:00 – 3:20PM

BREAK

3:20 – 5:00PM

SESSION 4: INNOVATION AND NEW TECHNOLOGIES

Moderated by Joseph Ciolino, MD

3:20 – 3:40pm Novel Adhesive Biomaterial for Repair and Regeneration of the Cornea Ahmad Kheirkhah, MD Instructor in Ophthalmology, Harvard Medical School Investigator, Schepens Eye Research Institute of Mass. Eye and Ear 3:40 – 4:00pm

anomedicine for Corneal Wound Healing N Alexander V. Ljubimov, PhD, FRSM Professor of Medicine, David Geffen School of Medicine at UCLA Professor of Biomedical Sciences and Neurosurgery Director, Regenerative Medicine Institute Eye Program Cedars-Sinai Medical Center

4:00 – 4:20pm

estoration of Corneal Transparency by Mesenchymal Stem Cells R Sunil Chauhan, DVM, PhD Associate Professor of Ophthalmology, Harvard Medical School Associate Scientist, Schepens Eye Research Institute of Mass. Eye and Ear

4:20 – 4:40pm

urgical Treatment of Ectatic Corneal Diseases—A Challenge S in our Days Joaquim Neto Murta, MD, PhD Professor of Ophthalmology Centro Hospitalar Universitário Coimbra, Faculty Medicine, Coimbra

4:40 – 5:00pm

Panel Discussion

5:00 – 6:00PM

J. WAYNE STREILEIN LECTURE

5:00 – 5:10pm Introduction to J. Wayne Streilein Lecture Ula Jurkunas, MD Associate Professor of Ophthalmology, Harvard Medical School Associate Scientist, Schepens Eye Research Institute of Mass. Eye and Ear Department of Ophthalmology Scholar, Mass. Eye and Ear 5:10 – 5:55pm J. Wayne Streilein Lecture: Trying to Tame a Chameleon—Our Efforts in Regulating Immunity in Corneal and Ocular Surface Disorders Reza Dana, MD, MSc, MPH Claes H. Dohlman Professor of Ophthalmology Director, Cornea & Refractive Surgery, Mass. Eye and Ear Senior Scientist, Mass. Eye and Ear, Schepens Eye Research Institute 5:55 – 6:10pm

Poster Abstract and Travel Award Winners Announcement

6:15 – 10:00pm

Cocktail Reception and Dinner at Liberty Hotel

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30th Biennial Cornea Conference

60 Years of Dr. Claes H. Dohlman’s Contributions to Corneal Science and Education Saturday, October 14, 2017 7:45 – 8:10am

Breakfast and Registration

8:10– 8:20am Welcome and Introduction

Reza Dana, MD, MSc, MPH (1995) Claes H. Dohlman Professor of Ophthalmology Committee on Immunology, Harvard Medical School Director, Cornea and Refractive Surgery Service, Mass. Eye and Ear Senior Scientist, Schepens Eye Research Institute of Mass. Eye and Ear 8:20 – 8:40am

laes H. Dohlman, MD, PhD C Professor of Ophthalmology and Chair Emeritus, Harvard Medical School Former Chief, Mass. Eye and Ear

8:40 – 9:40AM SESSION 1: ALUMNI I

Moderated by Peter Laibson, MD

8:40 – 8:52am A Cornea Specialty: The Early Days James Aquavella, MD (1963) Catherine E Aquavella Distinguished Professor of Ophthalmology University of Rochester Flaum Eye Institute 8:52 – 9:04am Remembrances of Dr. Dohlman Jules L. Baum, MD (1964) Professor of Ophthalmology, Emeritus Tufts-New England Medical Center 9:04 – 9:16am Reminiscing about a Corneal Fellowship and 53 Years of Friendship with Carin and Claes Peter Laibson, MD (1965) Director of the Cornea Service, Emeritus Wills Eye Hospital 9:16 – 9:28am Fifty Years after My Launch by Claes Henrik Dohlman, MD Stephen D. Klyce, PhD (1967) Adjunct Professor of Ophthalmology Icahn School of Medicine at Mount Sinai 9:28 – 9:40am

Following Instructions Michael A. Lemp, MD (1970) Clinical Professor of Ophthalmology Georgetown University and George Washington University

9:40 – 9:55am

Break

9:55 –11:20AM SESSION 2: ALUMNI II

Moderated by Kathryn Colby, MD, PhD

9:55 – 10:07am

laes H. Dohlman – One Woman’s Opinion C Deborah Langston, MD (1971) Professor of Ophthalmology, Emerita Harvard Medical School, Mass. Eye and Ear

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Harvard Department of Ophthalmology 10:07 – 10:19am Reflections in a Golden Eye Kenneth Kenyon, MD (1977) Clinical Professor of Ophthalmology Tufts University School of Medicine 10:19 – 10:31am

ppreciation and Admiration for Claes H. Dohlman, MD A Gary N. Foulks, MD, FACS (1978) Professor of Ophthalmology, Emeritus University of Louisville School of Medicine

10:31 – 10:43am

ungal Keratitis: Past, Present, and Future F Eduardo Alfonso, MD (1986) Kathleen & Stanley J. Glaser Chair in Ophthalmology Professor of Ophthalmology Director, Bascom Palmer Eye Institute Miller School of Medicine, University of Miami

10:43 – 10:55am Claes H. Dohlman: An Enduring Role Model on an Evolving Career Oliver Schein, MD, MPH (1988) Burton E. Grossman Professor of Ophthalmology Wilmer Eye Institute, Johns Hopkins School of Medicine 10:55 – 11:07am

op-10 Things I Learned from Claes Dohlman T Deborah Jacobs, MD (1992) Associate Professor of Ophthalmology (part-time) Harvard Medical School, Mass. Eye and Ear Medical Director, Boston Foundation for Sight

11:07 – 11:19am

echnology Advances in Presbyopia Correction T Roberto Pineda II, MD (1996) Associate Professor of Ophthalmology, Harvard Medical School Director, Keratorefractive Surgery Service Assistant Scientist, Mass. Eye and Ear

11:20 – 11:50AM

SESSION 3: PAST, PRESENT, AND FUTURE

11:20– 11:35am

laes H. Dohlman’s Legacy in Corneal Research C Ilene Gipson, PhD Professor of Ophthalmology, Harvard Medical School Ocular Surface Scholar and Senior Scientist, Schepens Eye Research Institute of Mass. Eye and Ear

11:35 – 11:50am KPro: Then and Now James Chodosh, MD, MPH David Cogan Professor of Ophthalmology in the field of Cornea and External Disease, Harvard Medical School Associate Director, Cornea and Refractive Surgery Service Senior Scientist, Mass. Eye and Ear 11:50AM – 12:55PM

LUNCH

12:55 – 2:10PM

SESSION 4: ALUMNI III

Moderated by Shahzad Mian, MD

12:55 – 1:07pm Rethinking Fuchs’ Dystrophy in the Era of Successful Descemet Stripping Kathryn Colby, MD, PhD (1998) Louis Block Professor of Ophthalmology & Visual Science Chair, Department of Ophthalmology & Visual Science University of Chicago Medical Center

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30th Biennial Cornea Conference 1:07 – 1:19pm Corneal VEGF Signaling: Angiogenic vs. Neurogenic Functions Mark Rosenblatt, MD, PhD (2005) Professor and Head, Department of Ophthalmology & Visual Sciences Illinois Lions/Charles I. Young Chair in Ocular Research Director, Corneal Regenerative Medicine Laboratory Illinois Eye and Ear Infirmary, University of Illinois at Chicago College of Medicine 1:19 – 1:31pm Lessons I Learned from Dr. Dohlman: The Vitreous is the Enemy of the Keratoprosthesis Victor Perez, MD (2000) Professor of Ophthalmology, Duke University Director, Ocular Surface Program, Duke University School of Medicine 1:31 – 1:43pm

volving Role of Eye Banks E Shahzad Mian, MD (2002) Professor, Ophthalmology and Visual Sciences Associate Chair, Education Kellogg Eye Center, University of Michigan

1:43 – 1:55pm

dvances in Study of Fuchs’ Endothelial Corneal Dystrophy with A Implications for Therapeutics Ula Jurkunas, MD (2006) Associate Professor of Ophthalmology, Harvard Medical School Associate Scientist, Schepens Eye Research Institute of Mass. Eye and Ear Department of Ophthalmology Scholar, Mass. Eye and Ear

1:55 – 2:07pm

oston Keratoprosthesis: Fresh versus Frozen Corneal Donor Carriers B Mona Harissi-Dagher, MD, FRCSC, DABO (2007) Associate Clinical Professor of Ophthalmology Chief of Cornea Service, Director of Cornea Fellowship Director of Ophthalmology Research Centre Hospitalier Université de Montréal

2:07-2:10pm

STRETCH BREAK

2:10 – 3:05PM

CLAES H. DOHLMAN LECTURE

2:10 – 2:20pm Introduction to Claes H. Dohlman Lecture

Reza Dana, MD, MSc, MPH (1995) Claes H. Dohlman Professor of Ophthalmology Director, Cornea and Refractive Surgery Service, Mass. Eye and Ear Senior Scientist, Mass. Eye and Ear, Schepens Eye Research Institute 2:20 – 3:05pm Claes H. Dohlman Lecture Science of Tearing Kazuo Tsubota, MD, PhD (1987) Professor and Department Chair Department of Ophthalmology Keio University School of Medicine 3:05 – 3:30 PM

CLOSING ACTIVITIES

3:05 – 3:15pm

Closing Remarks

3:15 – 3:30PM

GROUP PHOTO

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Harvard Department of Ophthalmology

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30th Biennial Cornea Conference

Participant Biographies and Abstracts

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Harvard Department of Ophthalmology

Friday, October 13, 2017 Reza Dana, MD, MSc, MPH Claes H. Dohlman Professor of Ophthalmology, Vice Chair for Academic Programs, Director, Harvard-Vision Clinical Scientist Development Program, Committee on Immunology, Harvard Medical School; Director, Cornea and Refractive Surgery Service, Mass. Eye and Ear; Senior Scientist and W. Clement Stone Scholar, Schepens Eye Research Institute of Mass.

Eye and Ear Dr. Reza Dana is an expert in corneal and ocular immunology. A graduate of Johns Hopkins and Harvard universities, his work focuses on the molecular and cellular mechanisms of ocular inflammation with applications in autoimmunity, transplantation, dry eye disease, stem cells, angiogenesis, and bioengineering. A Gold Fellow of the Association for Research in Vision and Ophthalmology (ARVO), he has authored more than 300 peerreviewed articles and more than 120 reviews and book chapters. His published work has been cited more than 19,000 times (h-index=72). He has received multiple awards, including the Physician-Scientist Award and Senior Investigator Award from Research to Prevent Blindness, the Cogan Award from ARVO, the Alcon Research Institute Award, and the Endre A. Balazs Prize from the International Society for Eye Research. He has also been named the Friedenwald Awardee for 2018 by ARVO. He is Associate Editor of Investigative Ophthalmology & Visual Science and is on the editorial board of multiple other journals, including Cornea, Scientific Reports (Nature Group), Ocular Surface, Ophthalmologica, and UpToDate Rheumatology. In addition to his basic laboratory investigations, he leads a translational research program at Mass. Eye and Ear that has received 10 IND permits from the U.S. FDA in the past decade. Dr. Dana has trained more than 110 fellows and graduate students from 32 countries in his laboratory to date, and in 2014 he received the A. Clifford Barger Excellence in Mentoring Award—the highest mentoring award bestowed at Harvard Medical School.

ABSTRACT Trying to Tame a Chameleon—Our Efforts in Regulating Immunity in Corneal and Ocular Surface Disorders This talk will provide an overview of some of the key pathophysiologic mechanisms of corneal inflammation, angiogenesis, and transplant immunity. Cornea-specific regulatory mechanisms, such as those expressed by the corneal epithelium and resident immune cells, which maintain immune homeostasis, will be emphasized. We will then review how chronic T cell-mediated autoimmunity and dry eye disease can subvert the normal homeostatic mechanisms that maintain immune quiescence by studying these factors in the context of corneal transplantation. Finally, we will provide overview of several novel strategies for augmenting immunoregulatory mechanisms, such as Treg expansion and use of tolerogenic antigen-presenting cells. Throughout, an emphasis will be placed on the plasticity and ever-changing phenotype of immune cells and the immune response, and if, in fact, it is possible to control a chameleon.

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30th Biennial Cornea Conference

Pablo Argüeso, PhD Associate Professor of Ophthalmology, Harvard Medical School; Senior Scientist, Schepens Eye Research Institute of Mass. Eye and Ear Dr. Pablo Argüeso earned his PhD in Biochemistry at the University of Valladolid in Spain. He conducts research in the fields of glycobiology and ophthalmology, focusing on the structure and function of glycoproteins on epithelial and endothelial cell surfaces. Dr. Argüeso has published more than 60 peer-reviewed manuscripts and book chapters in such journals as the Journal of Biological Chemistry, the Journal of Cell Science, and Nature Communications. He participates in a number of grant review panels and currently serves as a standing member of the Biology of the Visual System study section for the National Institutes of Health. A contributor to both national and international scientific societies, he has chaired the program committee (cornea section) for the Association for Research in Vision and Ophthalmology. He is currently the principal investigator for the P30 Core Grant for Vision Research and Director of the Morphology core at Schepens Eye Research Institute of Mass. Eye and Ear.

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Harvard Department of Ophthalmology

Noorjahan Panjwani, PhD Professor of Ophthalmology and Developmental, Molecular, and Chemical Biology, Director of Research, Department of Ophthalmology, Tufts University School of Medicine Dr. Noorjahan Panjwani is a glycobiologist. She earned her PhD in biochemistry of the eye at the University of Oxford, U.K. and completed her postdoctoral training in glycobiology at Harvard Medical School (Dr. Robert Spiro’s laboratory). Her research focuses on characterizing the role of carbohydrate-based recognition in the pathogenesis of wound healing and infections. Specifically, her group is investigating the function of cell surface receptors in the context of their glycosylation pattern and interactions with endogenous carbohydrate-binding proteins, galectins. Studies in her laboratory have shown that: (1) galectin-3 promotes corneal epithelial cell migration and re-epithelialization of corneal wounds by binding to complex N-glycans on a3b1 integrin and subsequently activating the a3b1 integrinRac1 signaling to promote lamellipodia formation (J Cell Sci. 2009;122: 3684-93), (2) galectin-3 promotes VEGF-A mediated angiogenesis by activating avb3 integrin and VEGFR2 (J Exp Med. 2010;207:1981-93; J Biol Chem. 2011;286:29913-21), (3) specific inhibitors of galectin-3 inhibit corneal neovascularization, as well as corneal and retinal fibrosis (Invest Ophthalmol Vis Sci. 2017;58:9-20); and (4) galectin-8 is a potent lymphangiogenic factor and that pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3 (Nat Commun.

2016;7:11302). Studies in her laboratory on the immunomodulatory role of galectins have demonstrated that: (1) local recombinant Gal-1 (rGal-1) treatment by subconjunctival injections significantly diminishes P. aeruginosa-mediated corneal inflammation through multiple mechanisms, including reduced proinflammatory Th17 cell response in the cornea, as well as local draining lymph nodes (J Immunol. 2013;190:6397-409), (2) Galectin-8 ameliorates murine autoimmune ocular pathology and promotes a regulatory T cell response by modulating IL-2 and TGFβ signaling (Immunol Cell Biol. 2015;Aug 18; PLoS One. 2015;Jun 30;10:6), and (3) galectin-8 knockout mice are resistant to P. aeruginosa keratitis.

ABSTRACT Galectin-8 Modulates Corneal Lymphangiogenesis and Infection Authors: Noorjahan Panjwani, Zhiyi Cao, Satoshi Sugaya, Maria J. Lopez, Victor G. Sendra, Pedram Hamrah & Wei-Sheng Chen Recent studies have demonstrated that the members of the galectin family of mammalian lectins play a critical role in regulating the pathology of a number of eye diseases, including corneal neovascularization, transplant rejection, corneal infection, and immunopathology of corneal infection. In the current study, we established a critical role of galectin-8 and carbohydrate-mediated recognition in the process of lymphangiogenesis. We demonstrated that galectin-8 expression is markedly upregulated in inflamed corneas; galectin-8 is a potent lymphangiogenic factor; and pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3. In addition, our studies revealed that (1) in the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection and (2) the severity of herpes simplex virus keratitis and P. aeruginosa keratitis is substantially reduced in Galectin-8-/- mice.

More importantly, subconjunctival injections with Gal-8N (the dominant negative inhibitor of galectin-8) significantly suppressed suture- and cautery-induced corneal lymphangiogenesis in mouse models of corneal inflammation. Additional studies revealed that activation of NLRP3 inflammasome pathway is required for clearance P. aeruginosa infection in mouse corneas, and that galectin-8 influences P. aeruginosa keratitis, most likely, by modulating the activation of NLRP3 inflammasome. These findings have broad implications for developing novel therapeutic strategies for conditions resulting from pathological lymphangiogenesis and immunopathology of corneal infections.

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30th Biennial Cornea Conference

Rajiv R. Mohan, MSc, PhD Ruth M. Kraeuchi Missouri Endowed Chair, Professor of Ophthalmology and Molecular Medicine, University of Missouri, Columbia Dr. Rajiv Mohan holds a Research Career Scientist position in the U.S. Department of Veterans Affairs at the Harry S. Truman Memorial Veterans’ Hospital in Columbia, Missouri. His research focuses on corneal gene therapy, nanomedicine, wound healing, glaucoma, and refractive laser surgery. Dr. Mohan’s laboratory has pioneered many tissue-selective localized gene therapies and nanomedicine approaches to combat scarring and angiogenesis in ocular tissues in vivo, which cause blindness in millions of people worldwide each year. Dr. Mohan’s research program is supported through grants from the NIH, the Veterans Health Administration, the American Glaucoma Society, and other agencies. He has authored more than 130 peer-reviewed journal articles, nine book chapters, and more than 300 scientific abstracts. Dr. Mohan has delivered numerous invited, distinguished, and keynote lectures and has provided vision research mentorship for more than 60 ophthalmology residents, clinical fellows, and both graduate and undergraduate students. Dr. Mohan has received numerous prestigious international honors, including the Newest and Most Innovative Research Award and Gold Fellow title from the Association for Research in Vision and Ophthalmology, the Mahatma Gandhi Pravasi Samman Award from India, and the Research Excellence Award from Zoetis. In addition, Dr. Mohan serves on several NIH and DoD study sections/panels and on the editorial board of multiple eye research journals, including Scientific Report, Translational Vision Science and Technology, PLoS One, Nanomedicine, Nanoscience Letters, and Ocular Pharmacology & Therapeutics. Dr. Mohan also serves on the advisory board of national and international research foundations, including the Missouri Medical Research Foundation, Narayan Netralaya Foundation, Aditya Jyot Foundation, and a number of professional eye and vision science research societies.

ABSTRACT Decorin+PEDF Gene Therapy Eliminates Corneal Neovascularization and Restores Vision in vivo Corneal neovascularization (CNV) is a leading cause of global blindness. Gene therapy, which provides long-term benefits and minimal adverse effects, is a novel approach to cure CNV. Decorin (DCN) and pigment epithelium-derived factor (PEDF) genes have been shown to regulate inflammation, fibrosis, and angiogenesis in the cornea. We tested the hypothesis that tissue-targeted and localized DCN+PEDF dual gene therapy delivered with AAV5 would eliminate CNV in rabbits in vivo by inducing selective apoptosis via the Fas-Fas ligand. A standard alkali injury-induced model of CNV in New Zealand White rabbits was used. Gene therapy was administered in vivo into the corneal stroma with a single topical administration of AAV5-DCN and PEDF titer (100μl; 5x1012vg/ml) either one day before or 30 minutes after alkali injury. Slit-lamp and stereo microscopy, H&E staining, and immunofluorescence were used to examine differential changes in the quantity, distribution, and length of vasculature present within the cornea, as well as keratocyte density, apoptosis, and overall ocular health. Immunoblotting and qPCR measured PEDF and Fas ligand expression. NIH ImageJ and Adobe Photoshop were used to quantify image data.

Tissue-targeted and localized AAV5-mediated DCN+PEDF gene therapy significantly reduced the number of vessels, vessel-density, and vessel-size (length and thickness) in rabbit corneas when given one day before injury (91-95%; p<0.001) or 30 minutes after alkali-injury (86-89%; p<0.001). The detection of significantly increased PEDF and Fas-ligand levels in conjunction with double-labeled CD31/lectin and TUNEL positive cells in DCN+PEDF delivered corneas suggested that CNV resolution is facilitated by apoptosis of endothelial cells from PEDF-induced Fas-Fas ligand signaling. Ongoing analysis of pro-and anti-angiogenic factors indicates that restoration of the physiological balance between factors is critical for corneal function and normal vision. AAV5-mediated DCN+PEDF gene therapy has the potential to cure CNV and restore vision in clinical settings. In vivo safety and toxicity studies are pending.

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Harvard Department of Ophthalmology

Michael Salter, MD, PhD, FRSC Senior Scientist and Chief of Research, The Hospital for Sick Children; Senior Scientist in the Program of Neurosciences & Mental Health, Professor of Physiology, University of Toronto Dr. Michael Salter earned an MD degree from the University of Western Ontario in 1982 and went on to obtain a PhD in Physiology from McGill in 1987. After postdoctoral training at Toronto Western and at Mt. Sinai hospitals, he joined the Research Institute of SickKids in 1990. From 1999 to 2009 Dr. Salter was the founding Director of the University of Toronto Centre for the Study of Pain. Dr. Salterâ&#x20AC;&#x2122;s main research focuses on synaptic physiology and how it relates to pain. His groundbreaking work has led to new paradigms about neuroplasticity and how synaptic transmission in the central nervous system is regulated by biochemical processes within neurons and by glial-neuronal interactions. His discoveries have broad implications for the control of cell-cell communication throughout the nervous system, and his work has regularly appeared in elite journals, including Nature, Science, Cell, Nature Medicine, and Neuron. Dr. Salter has a broad interest in neuroscience and his work is relevant to learning and memory, stroke-induced neuron death, epilepsy, and schizophrenia. As a distinct line of research, he and his collaborators discovered a previously unsuspected role for sensory neurons in the pathogenesis of diabetes and in the control of glucose homeostasis (Cell, 2006).

To facilitate the translation of his fundamental studies to the development of new therapies for humans, Dr. Salter is a founding scientist and actively involved in two startup biotech companiesâ&#x20AC;&#x201D;NoNO Inc. and Afference Therapeutics. Dr. Salter currently holds the Northbridge Chair in Paediatric Research. He has received numerous awards, including the E.B. Eastburn Award, the John Charles Polyani Prize in Physiology or Medicine, the Early Career Investigator Award of the Canadian Pain Society, and the Distinguished Career Investigator Award of the Canadian Pain Society. He was also an International Research Scholar of the Howard Hughes Medical Institute and is currently a Fellow of the Royal Society of Canada.

ABSTRACT Sex, Pain & Microglia Neuron-microglial interactions are increasingly recognized as key for physiological and pathological processes in the central nervous system. Microglia have been found to play a causal role in neuropathic pain behaviors resulting from peripheral nerve injury, and a core neuron-microglia-neuron signaling pathway has been elucidated. Within the dorsal horn, microglia suppress neuronal inhibition by a cascade that involves activating microglial P2X4 receptors that cause the release of brain-derived neurotrophic factor (BDNF). BDNF acts on trkB receptors, which leads to a rise in the intracellular Cl- concentration in dorsal horn nociceptive output neurons, transforming the response properties of these neurons. In addition to suppressing inhibition, peripheral nerve injury causes activity-dependent potentiation at dorsal horn glutamatergic synapses, which enhances nociceptive transmission. BDNF mediates the enhancement of synaptic NMDAR responses through activation of TrkB and the Src-family kinase, Fyn. We discovered that microglia-to-neuron signaling is not only critical for pain hypersensitivity after nerve injury, but also for the paradoxical hyperalgesic effect of morphine and other opioids. This core signaling pathway has been extensively characterized in studies using male mice. We recently discovered that microglia-neuron signaling is dispensable in female mice. Rather, pain hypersensitivity in female mice depends on the adaptive immune system, likely upon T cells. Despite this profound difference in cellular mechanisms, pain hypersensitivity in female mice is as robust as that in male mice. Taking into consideration sex differences in the spinal immune-neuronal signaling has important implications, ranging from diagnostics and therapeutics to prevention of chronic pain.

Funding: Supported by CIHR, Krembil Fdn, CRC, Anne and Max Tanenbaum Chairs, and Northbridge Chair.

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30th Biennial Cornea Conference

Mary Ann Stepp, PhD Professor of Anatomy and Regenerative Biology, George Washington School of Medicine and Health Sciences Before joining George Washington University School of Medicine and Health Sciences, Dr. Mary Ann Stepp conducted research at Schepens Eye Research Institute of Mass. Eye and Ear. In 1990, she and Ilene Gipson demonstrated that alpha 6 beta 4 integrin is a component of the hemidesmosomes. She also developed mouse models for the study of recurrent erosions and corneal stem cell deficiency, and she characterized whole-mount imaging methods to allow for high-resolution 3D image reconstructions of corneal goblet cell clusters, subbasal nerves, and intraepithelial nerve terminals. In addition, she has demonstrated the important roles of heparan sulfate syndecan-1 in mediating corneal epithelial and epidermal cell migration, phagocytosis, and axon targeting. She continues to be fascinated by corneal epithelial cells and is now studying how they function as glial cells to support the dense population of intraepithelial corneal nerves.

ABSTRACT The Corneal Epithelium and its Role in Maintaining the Sensory Nerves Authors: Mary Ann Stepp, Gauri Tadvalkar, and Sonali Pal-Ghosh The ophthalmic branch of the trigeminal ganglion contains somas of neurons that innervate the cornea.

These nerve fibers, known as intraepithelial corneal nerves (ICNs), provide sensory functions for the cornea and consist of subbasal nerves and intraepithelial nerve terminals. They project for several millimeters within the corneal epithelium without Schwann cell support. Corneal epithelial cells have evolved to function as glial cells to support the ICNs. Corneal epithelial cells activate in response to injury via mechanisms similar to those induced in Schwann cells during Wallarian degeneration. Corneal epithelial cells phagocytize distal axon fragments within hours of ICN crush wounds. ICNs shed their aged and damaged termini and continuously elongate to maintain their density, while corneal epithelial cells function like RPE cells to remove by phagocytosis shed axonal debris. Thus, the corneal epithelium and the intraepithelial corneal nerves depend on one another, and pathology in either one impacts both.

21

Harvard Department of Ophthalmology

Ula Jurkunas, MD Associate Professor of Ophthalmology, Harvard Medical School; Associate Scientist, Schepens Eye Research Institute of Mass. Eye and Ear Dr. Ula Jurkunas is a clinician scientist and surgeon in the Cornea and Refractive Surgery Service at Mass. Eye and Ear and Co-director of the Harvard Ophthalmology Cornea Center of Excellence. She conducts basic science and clinical research on Fuchs’ endothelial corneal dystrophy and stem cell transplantation of the ocular surface. Her main clinical areas of expertise are corneal endothelial dysfunction, femtosecond cataract surgery, refractive surgery, and lamellar keratoplasty, including DMEK and complex DSAEK. In addition, she teaches residents and fellows about corneal surgical procedures and the diagnosis and clinical management of corneal and refractive conditions.

Dr. Jurkunas was one of the first Harvard Ophthalmology faculty members selected to join the K12 Harvard Vision Clinical Scientist Development Program—which is funded by the NIH/NEI. As a K12 scholar, she conducted award-winning research on the pathophysiology of Fuchs’ endothelial corneal dystrophy, where she initiated efforts to link oxidative stress with the pathogenesis of Fuchs’ dystrophy. Today, she heads a fully staffed, RO1/NIH-funded laboratory that studies the mechanisms involved in the corneal endothelial degeneration seen in Fuchs’ dystrophy. Her studies focus on the role of oxidative stress in cell-extracellular matrix interactions, estrogen metabolism, DNA damage and repair, and mitochondrial biogenesis in Fuchs’ dystrophy.

Additionally, Dr. Jurkunas has pioneered the development of cultivated epithelial (stem) cell transplantation for the treatment of limbal stem cell deficiency in the United States. She was awarded a Production Assistance for Cellular Therapies (PACT) grant from the National Heart/Lung and Blood Institute of the NIH, which laid groundwork for the study of stem cell biology for the regeneration of corneal epithelium. The latter studies led to the translational development of stem cell therapy in corneal disorders. Dr. Jurkunas’ Investigational New Drug (IND) was approved, and she is beginning a Phase I/II study using stem cells to treat corneal blindness.

Additionally, with support from an NIH/NEI UG1 grant, she will perform the first in-human study using cultivated autologous limbal epithelial cells (CALEC) transplantation at Mass. Eye and Ear, in collaboration with Cell Manufacturing Facility at Dana-Farber Cancer Institute. Dr. Jurkunas has received many awards, including the ARVO Alcon Early Clinician-Scientist Research Award, Alcon Research Institute Award, American Academy of Ophthalmology Achievement Award, and, most recently, the Association for Research in Vision and Ophthalmology Foundation/Pfizer Ophthalmics Carl Camras Translational Research Award.

ABSTRACT Advances in Study of Fuchs’ Endothelial Corneal Dystrophy with Implications for Therapeutics The corneal endothelium is a cell layer that is susceptible to aging due to its post-mitotic arrest, high metabolic activity (from pumping ions), and lifelong exposure to ultraviolet light. Because the endothelium has minimal replicative capacity in vivo and is unable to replace its genome, it is prone to cumulative DNA damage acquired throughout a person’s lifetime. Fuchs’ endothelial corneal dystrophy (FECD) is a female-predominant, age-related disorder, in which the underlying genetic defects render the corneal endothelium genome susceptible to DNA damage, resulting in mitochondrial dysfunction. Currently, there are no medical therapies for FECD, and corneal transplantation is the only available treatment option. Our laboratory is investigating the roles of oxidative stress, oxidant-antioxidant imbalance, and mitochondrial dysfunction in the pathogenesis of FECD. Our studies detected an increase in mitochondrial and nuclear DNA damage, leading to the loss of mitochondrial membrane potential, decrease in ATP levels, and subsequent imbalance in mitochondrial fission and fusion mechanisms in FECD. Upregulation of mitophagy was noted to account for enhanced fission and loss of mitochondrial mass, activating caspase-dependent apoptosis. The findings of age-induced molecular changes underscore the degenerative aspects of FECD pathogenesis. Even though surgical therapies are effective in the treatment of FECD, they involve allogeneic transplantation. Therefore, the focus on developing novel cytoprotective and anti-aging therapies could provide alternative and safer options for FECD patients.

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30th Biennial Cornea Conference

Shivakumar Vasanth, PhD Instructor in Ophthalmology, Harvard Medical School; Investigator, Schepens Eye Research Institute of Mass. Eye and Ear Dr. Shivakumar earned his PhD from Kent State University-Cleveland Clinic, where he contributed significantly to the understanding of the interplay between STAT signaling pathways that are regulated by Polycystin-1—the protein that is mutated in autosomal dominant polycystic kidney disease. As a postdoctoral fellow at the National Institute of Environmental Health Sciences, National Institutes of Health, he continued his training in delineating the mechanism of proteins that localize to the primary cilia that lead to syndromic disorders collectively called ciliopathy. He transitioned into ophthalmology research as a Senior Research Associate in the Center for Human Disease Modeling at Duke University, North Carolina (headed by Nicholas Katsanis, PhD). During that time, he generated a knock-in mouse model for Fuchs’ endothelial corneal dystrophy (FECD) and used in vitro models to understand the causal mutations identified in patients with FECD. He further transitioned to continue his training in human genetics and the functional relevance of mutations identified in FECD in the laboratory of John D. Gottsch, MD, at the Wilmer Eye Institute, Johns Hopkins University. Dr. Vasanth analyzed trinucleotide repeat expansions in TCF4 in a large cohort of FECD patients and identified the threshold for causality for FECD. In addition, he used zebrafish as an in vivo model to understand the mechanistic details of lens development due to the loss of the downstream target of FOXE3, mutations that have been identified in patients with Peters anomaly. Dr. Vasanth is now interested in the studying DNA repair mechanisms that could be perturbed in FECD in the laboratory of Ula V. Jurkunas, MD. He is also the co-principal investigator of a research grant from Eversight Center for Eye and Vision Research that aims to further understand the relevance of oxidative stress-driven pathology of FECD to the genetic association signals identified in patients.

ABSTRACT Bridging the Gap in the Etiology of Fuchs’ Endothelial Cornea Dystrophy Authors: Shivakumar Vasanth, Takashi Miyai, Anne-Sophie Benischke, and Ula V. Jurkunas Fuchs’ endothelial corneal dystrophy (FECD) is an age-related and genetically complex disorder that affects approximately 4% of people over the age 40. The incidence in women is estimated to be three times higher than in men. FECD is characterized by a progressive, age-related loss of corneal endothelial (CE) cells that is accompanied by increased deposition of extracellular matrix, called guttae, that are visualized as excrescences on the Descemet membrane. These abnormalities drive the endothelial pathology that results in corneal swelling and loss of vision during the fifth and sixth decade of human life, thereby requiring corneal transplantation. FECD accounted for 49% of 32,221 endothelial keratoplasty (EK) procedures performed within the United States in 2016. Our laboratory at Schepens Eye Research Institute of Mass. Eye and Ear pioneered the work that demonstrated the role of oxidative stress in causing endothelial cell degeneration by detecting oxidation-induced nuclear (nDNA), as well as mitochondrial DNA (mtDNA) damage in FECD. A number of causal loci and associations have been identified for FECD, including the trinucleotide repeat expansion in TCF4 that accounts for two-thirds of the population in the United States. There is sufficient evidence that suggests aberrant expression of DNA repair genes play a role in the genetic modification of specific loci in repeat expansion diseases. In this study, we used normal donor corneal endothelial tissue and FECD endothelial keratoplasty specimens stratified by their genotypes as either expanded or not expanded for TCF4. We performed PCR arrays to analyze the differential gene expression of 84 DNA repair genes that could be aberrantly expressed in FECD, and specifically, in patients who harbor an expansion of TCF4. We identified multiple genes that are differentially expressed that play a role in mismatch, base excision, and double strand break repair processes that may explain the increased DNA damage previously identified in FECD. More importantly, the upregulation of a specific mismatch repair protein in FECD specimens that harbor an expansion strengthens the idea of aberrant mismatch repair in FECD leading to expansion of trinucleotide repeats in TCF4. This study, aims to bridge the gap that exists in the understanding the effect of DNA repair in repeats expansions.

23

Harvard Department of Ophthalmology

Joseph Bonanno, OD, PhD Professor and Dean, Indiana University School of Optometry Dr. Joseph Bonanno earned his BA in Biology from the University of Pennsylvania (1975), MS in Molecular Biology (1977), OD (1981), and PhD in Physiological Optics (1987) from the University of California, Berkeley.

Dr. Bonanno completed a Primary Care Residency at the Eye InstitutePennsylvania College of Optometry (1982) and was Director of the Contact Lens Service at Wills Eye Hospital (1982-83). Following his PhD, he completed postdoctoral work at the Louisiana State University Department of Ophthalmology (1987–88) and the University of California, Berkeley, Department of Physiology-Anatomy (1988–89). He then joined the faculty at UC Berkeley School of Optometry in 1989 as Assistant Professor, where he attained the rank of Professor in 1997. He joined the faculty of Indiana University School of Optometry in July 1998 as Professor of optometry.

Dr. Bonanno’s research interests include the corneal endothelial pump, contact lens-induced corneal physiological effects, and hypoxia preconditioning protection. For 26 years, he has received continuous support from the NEI to study the mechanisms of the endothelial pump. He served on the National Advisory Eye Council from 2009 to 2012 and was named a fellow of ARVO in 2009. Dr. Bonanno has served various administrative duties at IU Optometry and was appointed Dean of the school in 2010.

ABSTRACT Endothelial Dystrophy in the Slc4a11 Knock Out Mouse Authors: Joseph A. Bonanno, Wenlin Zhang, Moonjung Choi, Shimin Li, and Diego Ogando SLC4A11 mutations are a signature feature of congenital hereditary endothelial dystrophy (CHED) and some forms of Fuchs’ dystrophy. The Slc4a11 knock-out mouse shows corneal edema at eye opening.

At 12 weeks, endothelial cell density is not significantly different from wild-type; however, at 40 weeks it is reduced by 15%. Corneal [lactate] is twice that of wild-type, consistent with the endothelial pump being comprised of Na+: K+-ATPase, carbonic anhydrases, Na+:2HCO3- cotransport and other cell buffering mechanisms acting in concert to facilitate the efflux of water coupled to lactic acid. Other hallmarks of endothelial dystrophy, such as oxidative stress, mitochondrial disruption, and increased autophagy, were also present in the mouse corneal endothelium. While mutations in Col8A2 and TCF4 produce endothelial dystrophy via secondary toxic mechanisms, loss of Slc4a11 function directly leads to endothelial dysfunction. Slc4a11 codes for a membrane protein that acts as an H+ channel or NH3:2H+ cotransporter. Ammonia is a product of amino acid catabolism, occurring mostly within mitochondria. In human and mouse corneal endothelial cells, we found that TCA cycle intermediates were predominantly derived from glutamine, whereas in the Slc4a11 knock-out cells, glucose was preferred. Cell lines derived from the knock out also showed increased mitochondrial oxidative stress, impaired mitochondrial membrane potential, greater sensitivity to glutamine induced apoptosis, and reduced ability to generate ATP. These deficits could be partially offset by inhibition of glutaminase, the enzyme that catalyzes the conversion of glutamine to glutamate, producing ammonia. Supplementation with dimethyl-alphaketoglutarate was the most effective in reducing NH3 production and reversing mitochondrial dysfunction.

We conclude that the ammonia sensitive H+ permeability of Slc4a11 plays an important role in ammonia detoxification within mitochondria. Loss of Slc4a11 activity leads to mitochondrial dysfunction and reduced ATP generation, which ultimately slows the endothelial pump.

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30th Biennial Cornea Conference

Shigeru Kinoshita, MD, PhD Professor and Chair, Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine Clinician scientist Dr. Shigeru Kinoshita graduated from Osaka University Medical School in 1974, and has served as the Professor and Chair of Ophthalmology at Kyoto Prefectural University of Medicine since 1992. After stepping down from the Chair of Ophthalmology in March 2015, he was elected the Professor and Chair of Frontier Medical Science and Technology for Ophthalmology at Kyoto Prefectural University of Medicine in April 2015.

He continues to work as a distinguished clinician scientist.

In the early 1980s at Harvard Medical School, he collaborated with Dr. Richard A. Thoft to establish the concept of centripetal movement of the corneal epithelium. His groundbreaking work has shed new light on the importance of the limbal epithelium. His series of findings has had an enormous impact on this subject and has afforded much insight, ultimately contributing to the development of the corneal stem cell theory set forth

by Tuen-Tien Sun. Based on these concepts, Dr. Kinoshita developed a new surgical procedure for in vivo corneal epithelial transplantation that led to epithelial stem cell transplantation for ocular surface rehabilitation. Over the past 30 years, his primary interest has been focused on the translational research of new therapeutic modalities for severe corneal diseases. Following this path, his group recently established the system of cultivated mucosal epithelial stem cell transplantation for severe ocular surface disorders, such as Stevens-Johnson syndrome and chemical injury, and cultivated corneal endothelial cell transplantation for bullous keratopathy. His group also proved the clinical efficacy of Rho-associated protein kinase (ROCK)inhibitor topical application for partial endothelial dysfunction, aiming at the development of novel therapies for corneal endothelial dysfunction.

Named an ARVO Gold Fellow in 2009, Dr. Kinoshita has received numerous awards, including the 1999 Alcon Research Institute Award, the 2008 Castroviejo Medal from the Cornea Society, the 2010 Claes H. Dohlman Conference Address of the Tear Film & Ocular Surface Society, the 2011 Doyne Memorial Lecture at the Oxford Ophthalmological Congress, the 2011 Schepens Eye Research Institute of Mass. Eye and Ear Alumnus Award, the 2015 Charles D. Kelman Innovator Award from the American Society of Cataract and Refractive Surgery, the 2016 Friedenwald Award from ARVO, the 2017 Coster Lecture at the Australian and New Zealand Cornea Society, and the 2017 David Easty Lecture at the Bowman’s Club. He served as an ARVO Program Committee Member in the Cornea Section between 1996 and 1999 and an ARVO Trustee of the Cornea Section between 2006 and 2011.

ABSTRACT Future Realities in Corneal Endothelial Regenerative Medicine Understanding the biological and immunological characteristics of human corneal endothelial cells (CECs) is an essential key to establishing new treatment strategies for corneal endothelial dysfunction, such as Fuchs’ endothelial corneal dystrophy, intraocular-surgery-related bullous keratopathy, graft failure, etc. In this direction, our research group has been developing a novel ‘cell-injection therapy’ that involves the injection of cultured human CECs into the anterior chamber. For this purpose, non-proliferative corneal endothelial cells from donated corneas have been induced to proliferate, without inducing cell state transition (CST). Our findings in 35 clinical research cases have shown this approach to be promising.

However, in order to apply this novel approach to actual clinical practice, several key safety issues must be addressed and assured from the aspect of regulatory science. It is our great hope that this procedure will receive official governmental approval based on the accumulated data of safety and efficacy aspects in clinical trial started in 2017.

25

Harvard Department of Ophthalmology

Anthony Aldave, MD Professor of Ophthalmology, Walton Li Chair in Cornea and Uveitis, Chief, Cornea and Uveitis Division, Director, Cornea and Refractive Surgery Fellowship, UCLA Stein Eye Institute Dr. Anthony J. Aldave, MD, earned his medical degree from the University of Texas Medical Branch in Galveston. He completed his residency training in ophthalmology at the Wills Eye Hospital, Thomas Jefferson University, in Philadelphia, Pennsylvania. Dr. Aldave then completed a two-year Cornea, Uveitis and Refractive Surgery Fellowship at The University of California, San Francisco/Francis I. Proctor Foundation. He joined the Stein Eye Institute in July 2002 as a full-time member of the Cornea and Refractive Surgery Services.

Dr. Aldave has received numerous honors during his career, including the Alpha Omega Alpha Scholarship Award for graduating first in his medical school class; the Heed Ophthalmic Foundation Fellowship; the American Ophthalmological Society-Knapp Testimonial Fund Fellowship; the first Claes Dohlman Society Award; and the Achievement, Secretariat, and Senior Achievement Awards from the American Academy of Ophthalmology.

In addition to his busy clinical practice, Dr. Aldave directs the Corneal Genetics Laboratory at the Stein Eye Institute. Dr. Aldave’s NIH-funded laboratory research focuses on the molecular genetics of the corneal dystrophies—a group of inherited disorders that affect corneal clarity and constitute one of the primary indications for corneal transplantation. Recognized as a leader in his chosen field of scientific investigation, Dr. Aldave has authored more than 100 peer-reviewed scientific publications; has been invited to give more than 300 presentations at local, national, and international meetings; and serves as a scientific reviewer for 18 ophthalmology and genetics journals.

ABSTRACT Elucidating the Genetic Basis of Posterior Polymorphous Corneal Posterior polymorphous corneal dystrophy (PPCD) is a dominantly inherited disorder of the corneal endothelium that has been linked to truncating mutations in the zinc finger E-box binding homeobox 1 gene (ZEB1) on chromosome 10 (PPCD3) and promoter mutations in the ovo-like zinc finger 2 gene (OVOL2) on chromosome 20 (PPCD1). While the genetic basis of PPCD1 and PPCD3 have been identified, the majority of PPCD cases are still genetically undetermined. OVOL2 and ZEB1 encode transcription factors that regulate prenatal development and are involved in malignant transformation. In addition, OVOL2 and ZEB1 form a mutually inhibitory circuit that regulates cellular plasticity (e.g., stem cells, cancer) and maintains distinct cell states. While ZEB1 truncating mutations are associated with PPCD, ZEB1 missense mutations are associated with Fuchs’ endothelial corneal dystrophy (FECD), which has also been associated with an intronic SNP and an intronic trinucleotide repeat in TCF4. It is hypothesized that ZEB1 truncation leads to haploinsufficiency and to PPCD (an early-onset disease), while ZEB1 missense mutations lead to altered functional properties—the cumulative effect of which leads to FECD (a late-onset disease). A greater understanding of how this genotype-phenotype difference results in PPCD and FECD will provide insight into the molecular basis of each dystrophy.

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30th Biennial Cornea Conference

Natalie Afshari, MD, FACS Professor of Ophthalmology, Stuart I. Brown M.D. Chair in Ophthalmology in Memory of Donald P. Shiley, Chief, Division of Cornea and Refractive Surgery, Vice Chair of Education, Shiley Eye Institute at UC San Diego Health Before joining Shiley Eye Institute, University of California, Dr. Natalie Afshari was Professor of Ophthalmology and Director of Centers of Excellence at the Duke University Eye Center. She earned her medical degree from Stanford University and completed her residency training at Harvard Ophthalmology.

She then completed a two-year fellowship in cornea, refractive surgery, and external disease at Mass. Eye and Ear. Dr. Afshari received the Senior Achievement Award and the Secretariat Award from the American Academy of Ophthalmology and has been named a Gold Fellow of the Association for Research in Vision and Ophthalmology (ARVO).

She received the inaugural Top-Ten Women in Medicine award from Triangle News, the Women Who Mean Business award from the San Diego Business Journal, and the Teacher of the Year award at the Duke University Eye Center.

She has also been recognized as one of the Best Doctors in America in each listing for the past decade, and was included on the U.S. News & World Report’s Top Doctors List.

Dr. Afshari is the Co-editor of the two-volume cornea book titled, Principles and Practice of Cornea. She is also on the editorial boards of Investigative Ophthalmology and Visual Science and the American Journal of Ophthalmology. She has previously served on the EyeNet editorial board, Basic and Clinical Science Cornea text book committee, and the American Academy of Ophthalmology council representing the American Society of Cataract and Refractive Surgery. She was Co-chair of the cornea program committee for ARVO and Co-director of Cornea Subspecialty Day for the American Academy of Ophthalmology. She is currently the Chair of the American Society of Cataract and Refractive Surgery FDA Committee. Her NIH research grant supports her Fuchs’ dystrophy research, and she investigates the intricacies of endothelial keratoplasty and regeneration of cornea.

ABSTRACT Fuchs’ Dystrophy and Corneal Endothelium from Genetics to Stem Cells and Regeneration Authors: Natalie A. Afshari MD, et al.

Fuchs’ endothelial corneal dystrophy (FECD) is the most prevalent indication for corneal transplantation in the United States. This talk will cover the genetics of Fuchs,’ as well as the regeneration of the corneal endothelium through differentiation of human pluripotent stem cells (PSCs) into corneal endothelial cells.

A genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry was performed. This GWAS was followed by replication and meta-analysis for a total of 2,075 cases and 3,342 controls. For regeneration of cornel endothelium, the fate of stem cells was restricted to the eye field-like state, and the cells became eye field stem cells (EFSCs). In the second phase, PSC-derived EFSCs were further directed toward either neural crest lineage or retinal lineage. The corneal endothelial cells were directly induced from ocular neural crest stem cells (NCSCs) by suppressing TGF-β and ROCK signaling. In the GWAS study, we identified three significant novel loci (Po5 10 8): KANK4 rs79742895, LAMC1 rs3768617, and LINC00970/ATP1B1 rs1200114. We also observed a strong signal from the established TCF4 locus. Furthermore, we developed a novel, small-molecule-based approach to derive human corneal endothelial cells from PSCs via ocular lineage specification. The NCSC-derived corneal endothelial cells expressed typical corneal endothelial cell markers, such as N-Cadherin and Na+/K+-ATPase. We found three new genes in FECD. Each of those genes plays a possible role in the pathogenesis: KANK4 and LAMC1 may be involved in maintaining the endothelial cell layer, while ATP1B1 likely functions in fluid transport. These findings may shed light on the biochemical pathogenesis of FECD. Additionally, EFSC-derived NCSCs could serve as an immediate source cell for rapid corneal endothelial cell induction.

27

Harvard Department of Ophthalmology

Andrew Taylor, PhD Associate Dean for Research, Professor and Director of Research, Ophthalmology, Boston University School of Medicine Dr. Taylor earned his PhD from the Department of Microbiology at Ohio State University and completed a postdoctoral fellowship with Scott Cousins, MD, and J. Wayne Streilein, MD, at the University of Miami School of Medicine.

In 1993, he joined the Harvard Ophthalmology faculty. In 2010, Dr. Taylor joined the faculty of the Department of Ophthalmology, and the Immunology Training Program of Boston University School of Medicine. He was appointed in 2015 the School of Medicineâ&#x20AC;&#x2122;s Associated Dean for Research. Dr. Taylor has served on several NIH, DoD, and other domestic and international review panels, and he is currently a member of the Diseases and Pathology of the Visual System NIH review panel. Dr. Taylor regularly reviews manuscripts for multiple immunology and vision science journals, and is on the editorial board for Ocular Immunology and Inflammation. He is a member of the board of directors of the Streilein Foundation for Ocular Immunology (a nonprofit organization promoting research, training, and mentorship in immunology). Dr. Taylor is an active member and Gold Fellow of the Association for Research in Ophthalmology and Vision (ARVO). Dr. Taylor leads a research program of excellence that is internationally recognized for its work into the molecular mechanisms of ocular immune privilege and immunobiology of the eye. His research demonstrates the importance of neuropeptides in regulating adaptive and innate immunity, the alternative activation of retinal microglia and macrophages, the mechanisms of melanocortin regulation of immunity, and how to adapt the mechanisms of ocular immune privilege suppress ocular autoimmune disease, transplantation graft rejection.

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30th Biennial Cornea Conference

Pedram Hamrah, MD Associate Professor of Ophthalmology, Director, Center for Translational Ocular Immunology, Tufts University School of Medicine Dr. Pedram Hamrah is a clinician scientist who focuses on corneal immunology, ocular imaging (immuno-imaging), ocular surface disease, and corneal neuropathic pain. He earned his medical degree from the University of Cologne in Germany and completed his residency in ophthalmology at the University of Louisville, where he was elected Chief Resident during his last year. He completed fellowships in Ocular and Transplantation Immunology at Mass.

Eye and Ear, and in Ocular Immunology at the University of Louisville, as well as a two-year clinical fellowship in Cornea, External Diseases and Refractive Surgery at Mass. Eye and Ear. He is currently on the faculty at the Department of Ophthalmology at Tufts University School of Medicine, where he is the Director of the Center for Translational Ocular Immunology. In addition, he is a faculty member for the Immunology, Neuroscience, and Cell, Molecular and Developmental Biology graduate programs at the Sackler School of Graduate Biomedical Sciences at Tufts. Throughout his career, he has focused on discovery, patient care, and teaching.

Dr. Hamrah has been directing both strong laboratory science and prolific clinical research programs, currently holding three NIH grant awards, in addition to numerous foundation and industry grants. Prior to joining Tufts, he was on the faculty at Harvard Ophthalmology and a member of Mass. Eye and Ear’s Cornea Service for seven years, where he was also the Founder and Director of the Ocular Surface Imaging Center. Dr. Hamrah currently serves on over a dozen editorial boards, is the Section Editor for The Ocular Surface and Eye and Assistant Editor at Ocular Immunology and Inflammation. Dr. Hamrah has been continuously serving on the Members-in-Training, Professional Development and Education and the Ethics and Regulations in Human Research Committees at ARVO since 2003 and is currently an ARVO Gold Fellow.

ABSTRACT Corneal Plasmacytoid Dendritic Cells—The New Cells on the Block Plasmacytoid dendritic cells (pDC), a distinct type of bone marrow (BM)-derived cell, play an important role in linking innate and adaptive immune responses and are implicated in the induction of tolerance to transplanted tissues or tumors. Our data demonstrate that pDC, which typically present in lymphoid organs, reside in the cornea and retina. To date, despite the well-studied functions of leukocytes in angiogenesis, the role and source of endogenous angiostatic molecules, which preserve corneal angiogenic privilege or prevent retinal neovascularization, remain elusive. Our studies have shown that depletion of pDCs is accompanied by abrupt and severe breakdown of angiogenic privilege of the cornea. Subsequent repopulation of corneal pDC leads to regression of corneal neovascularization.

Similarly, increased neovascularization is observed after intrastromal suture placement in pDC-depleted corneas as compared to controls. In pDC-depleted corneas, relative endostatin and thrombospondin-1 mRNA levels are decreased after pDC depletion. Direct expression of angiostatic molecules is shown by flow cytometry, which shows that both endostatin and thrombospondin-1 co-stained with pDCs in both naïve and suture-induced inflamed corneal single cell suspensions. Local adoptive transfer of green fluorescent protein (GFP)+ pDC with epithelial debridement and tissue glue application to the cornea leads to diminished neovascularization induced by suture placement and demonstrate the angiostatic effect of corneal pDC. Similarly, direct co-cultures of pDCs with vascular endothelial cells confirm their angiostatic functions. Thus, pDCs exhibit angiostatic functions by actively secreting anti-angiogenic molecules and are necessary for preserving corneal angiogenic privilege as well as limiting retinal neovascularization in pathologic conditions. Cell-based approach through utility of pDCs could potentially introduce new therapeutic avenues in various pathologic conditions in order to suppress ocular angiogenesis.

29

Harvard Department of Ophthalmology

Jaya Rajaiya, PhD Assistant Professor of Ophthalmology, Harvard Medical School; Assistant Scientist, Mass. Eye and Ear Human adenoviruses within species D—specifically types 8, 37, 53, 54, 56, and 64—cause epidemic keratoconjunctivitis (EKC), which is an acute, highly contagious, and often severe keratoconjunctivitis, associated with prolonged and/or recurrent corneal stromal inflammation. To date, no specific therapy exists for adenovirus infections. Dr. Rajaiya’s PhD work focused on developing a recombinant vaccine for tuberculosis and other mycobacterial diseases. She participated in a World Health Organization student exchange program and worked at the Medical Research Council in Hammersmith, London. During this time she received an Award to Inventors and procured a patent that was licensed to Inflazyme, Canada. Her postdoctoral training was in basic molecular biology and focused on transcription factors and signaling molecules in gene expression. Her current work applies her expertise in molecular biology and infectious disease pathogenesis to questions in adenoviral pathogenesis. She has also expanded her expertise to cellular and nuclear signaling that leads to inflammation upon adenoviral infection. Dr. Rajaiya trains and directs postdoctoral fellows, students, and technicians with their work. Her current studies are focused on the molecular biology of viral entry, in particular the roles of caveolin and dynamin in adenovirus entry and nuclear trafficking in corneal cells. She is also involved in a large study of adenovirus “omics.” This latter project seeks to elucidate the viral transcriptome and proteome of human adenoviruses most closely associated with corneal infection.

Her recently published work shows that recombination amongst adenoviruses can create new viruses with tropism for the cornea. Her goal is to identify the specific loci of recombination in order to understand the mechanism(s) of recombination and predict the emergence of new EKC pathogens in the future.

ABSTRACT Pivotal Role of Dynamin in Adenovirus Trafficking in Corneal Cells Authors: Jaya Rajaiya, Ji Sun Lee, Ashrafali Mohamed Ismail, Jeong Yoon Lee, Emma C. Materne, and James Chodosh Microtubules play a critical role in trafficking adenoviruses. Here, we report that dynamin 2 negatively regulates adenoviral trafficking through its influence on microtubule dynamics. Dynamin 2 knockdown in corneal fibroblasts that were subsequently infected with adenovirus resulted in the accumulation of acetylated tubulin, increased Src activation, movement of microtubule organizing centers (MTOCs) closer to cell nuclei, increased virus in the cytosol (rather than the endosome), and enhanced nuclear delivery of viral DNA. Overexpression of dynamin 2 generated strictly opposite effects. Dynamin 2 knockdown also led to a greater association between Nup 358/214 and virus as compared to overexpression. Inhibition of endosomal acidification had no effect on adenovirus infection, while inhibition of actin and microtubule polymerization reduced viral gene expression. Most interestingly, dynamin 2 knockdown reduced cytokine expression upon infection, while dynamin 2 overexpression increased cytokine expression. These results are consistent with a negative role for dynamin 2 in adenovirus infection, and further implicate novel functions of this pleiotropic cellular molecule. Dynamin 2 and microtubule acetylation and organization play interdependent and contrary roles in adenoviral trafficking in an infected corneal fibroblast. The reduction of cellular innate immune responses to infection in cells in which dynamin 2 is overexpressed suggests potential clinical applications to adenovirus infection.

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30th Biennial Cornea Conference

Claus Cursiefen, MD Chairman and Professor, Department of Ophthalmology, University of Cologne, Germany Dr. Cursiefen presented Lymphangiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye at the DFG Research Unit FOR 2240.

He is Action Chair of the European Cooperation in Science and Technology EU COST) Action BM1302: Joining Forces in Corneal Regeneration. He is also a member of the DFG Study Board Neuroscience (Fachkollegium), the Board of the German Ophthalmological Society (DOG), and the German National Academy of Science Leopoldina.

Dr. Cursiefen completed his medical studies at the Universities of Regensburg/ Germany, Würzburg/Germany, King’s College/London, University of Dundee/ St. Andrews/Scotland and Malta. He completed his MD thesis at the Department of Anatomy, University of Würzburg and his residency at the Department of Ophthalmology in Erlangen/Germany. He subsequently completed a postdoctoral fellowship at Schepens Eye Research Institute of Mass. Eye and Ear. Dr. Cursiefen’s research focuses on the mechanisms of corneal hem- and lymphangiogenesis and their implications for corneal transplant immunology, as well as modern lamellar corneal transplant surgery.

His clinical interests include novel lamellar surgical techniques in corneal transplantation; ocular surface reconstruction and dry eye disease; and cataract, glaucoma, and refractive surgeries.

ABSTRACT Immunological Graft Rejections After DMEK Authors: Claus Cursiefen, Bjoern Bachmann, Felix Bock, Deniz Hos The researchers aimed to analyze the incidence and clinical course of graft rejection episodes after Descemet membrane endothelial keratoplasty (DMEK). They conducted a retrospective analysis of a consecutive, interventional case series, which included 1,000 eyes that underwent DMEK from July 2011 through August 2015 at the Department of Ophthalmology, University of Cologne. All cases with follow-up of at least 1 month were included (mean follow-up, 18.5 months). Patients with a graft rejection episode were followed up for 1 additional year. Outcome measures included: incidence of graft rejection, best spectacle-corrected visual acuity (BSCVA), central corneal thickness (CCT), endothelial cell density (ECD), and need for regraft. Nine hundred five cases met the inclusion criteria. A graft rejection episode developed in 12 patients (estimated probability of rejection at 1 year, 0.9%; at 2 years, 2.3%; at 4 years, 2.3%). At the time of rejection, 9 of 12 patients had stopped corticosteroids. Five patients were symptomatic and 7 did not note the rejection episode. Intensified topical corticosteroid therapy was started immediately after rejection was diagnosed. Two eyes decompensated and required a regraft, whereas the remaining 10 eyes required no regraft (BSCVA, 0.27±0.28 logarithm of the minimum angle of resolution [logMAR]; CCT, 554.1±39.1 μm at last visit before rejection vs. BSCVA, 0.21±0.15 logMAR; CCT, 540.0±15.0 μm 3 months after rejection). One year after the rejection episodes, BSCVA and CCT in these eyes remained unchanged when compared with the last visit before rejection (BSCVA, 0.15±0.11 logMAR; CCT, 533.8±26.0 μm).

Significant changes were observed for ECD values (1741±274.5 cells/mm2 at last visit before rejection vs.

1356±380.3 cells/mm2 after 3 months [P = 0.04] and 1290±359.0 cells/mm2 after 1 year [P = 0.01]). The risk for graft rejection after DMEK is low, and an even smaller minority requires a regraft. After intensified local corticosteroid therapy, most patients show stable visual acuity and CCT, although ECD decreases.

The occurrence of immune reactions up to 2 years after surgery predominantly in patients not receiving corticosteroids supports the prolonged use of corticosteroids after DMEK.

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Harvard Department of Ophthalmology

Susmit Suvas, PhD Associate Professor, Departments of Ophthalmology, Anatomy & Cell Biology, and Immunology and Microbiology, Wayne State University School of Medicine Dr. Suvas earned his doctorate in Cellular Immunology from the Institute of Microbial Technology (IMTECH), a CSIR institute in India. During his PhD, he worked on the concept of reverse signaling through B7 molecules and published his results in J Biol Chem. (2002). This was the first demonstration of signaling through B7 molecules in B cell lymphoma. This paper has been cited more than 200 times. After graduating, he joined the laboratory of Barry Rouse at the University of Tennessee, as a postdoctoral researcher.

During his training as a viral immunologist, he studied the role of CD4+CD25+ regulatory T cells in viral infection. His group was the first to report that administration of anti-CD25 (IL-2 receptor alpha) antibody enhances memory CD8 T cell pool in herpes simplex virus-1 infected mice. Those results were published in J Exp Med (2003), and the manuscript has been cited more than 500 times. In 2007, he joined the Department of Biology at Oakland University as an Assistant Professor.

As an independent investigator, he initially dabbled in the area of inhibitory receptors and regulatory T cells.

In 2010, he became interested in understanding the role of neuro-immune axis in the pathogenesis of HSK. That same year he was awarded R21 funding from the NEI to study neuropeptides and herpes simplex virus-1 infection. In 2013 he received his first R01 grant from the NEI. In 2014, he joined Wayne State University School of Medicine as an Associate Professor in the Departments of Ophthalmology and Anatomy & Cell Biology. Currently, his laboratory is ascertaining the role of neuropeptides in the pathogenesis of HSK and the ocular surface homeostasis. He has published his findings on neuropeptides and HSK in several peer-reviewed journals, such as Investigative Ophthalmology and Visual Science and the Journal of Immunology.

ABSTRACT Neuropeptide Mediated Regulation of the Ocular Surface Homeostasis and Herpes Stromal Keratitis Authors: Subhash Gaddipati, Pushpa Rao, Andrew Jerome, and Susmit Suvas Earlier, we reported that blocking neurokinin-1 (NK1R)â&#x20AC;&#x201D;the highest affinity receptor of substance P neuropeptideâ&#x20AC;&#x201D;in an ongoing HSV-1-induced corneal inflammation reduces the severity of herpes stromal keratitis (HSK). However, when mice lacking functional NK1R were infected with corneal HSV-1 infection, they developed more severe HSK than wild type C57BL/6 mice. While trying to determine the underlying mechanism, we found that the ocular surface of un-manipulated NK1R-/- mice was different from control C57BL/6 mice. This involved an excessive desquamation of apical corneal epithelial cells, an increased epithelial cell density, but decreased epithelial cell size in NK1R-/- mice. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells, and a reduced volume of basal tears. However, a massive accumulation of CD11b+ myeloid cells was seen near the limbal area of the corneas from NK1R-/- mice. An increased myelopoiesis was also seen in the bone-marrow of NK1R-/mice. Our results showed that accumulation of CD11b+ cells at the limbal area of NK1R-/- mice occurs at P14 but not at P7 stage post-birth. After corneal HSV-1 infection, the numbers of CD11b+ cells and neutrophils infiltrating the infected corneas was significantly higher in NK1R-/- than C57BL/6 mice. This was associated with an increased viral load in infected corneas of NK1R-/- mice. As a result, the number of IFN-g secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R-/- mice was much higher than C57BL/6 mice. An increased number of CD4 T cells and mature neutrophils in inflamed corneas of NK1R-/- mice was associated with an early development of severe HSK. Collectively, our results show that under steady state NK1R signaling is important to maintain the homeostasis of ocular surface, but in an ongoing corneal inflammation NK1R signal enhances the inflammatory events.

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30th Biennial Cornea Conference

Joseph B. Ciolino, MD Assistant Professor of Ophthalmology, Harvard Medical School; Henry Freeman Allen Cornea Scholar, Investigator, Massachusetts Eye and Ear Dr. Joseph Ciolino is a clinician scientist at Massachusetts Eye and Ear. His research has been supported by the U.S. Department of Defense, the National Institutes of Health, and other funding organizations. He has also served as an advisor and medical monitor for more than 25 ophthalmic clinical trials.

Dr. Ciolinoâ&#x20AC;&#x2122;s primary research focuses on improving outcomes of the Boston keratoprosthesis and developing a drug-eluting contact lens. The contact lens has demonstrated sustained release of therapeutic drug levels for up to one month and efficacy in glaucomatous monkeys. This technology has been covered by popular media, including CNN, The Economist, Reuters News Service, and Scientific American.

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Harvard Department of Ophthalmology

Ahmad Kheirkhah, MD Instructor in Ophthalmology, Harvard Medical School; Investigator, Schepens Eye Research Institute of Mass. Eye and Ear Dr. Ahmad Kheirkhah is a cornea and ocular surface specialist who has been an Instructor at Harvard Medical School and an Investigator at Massachusetts Eye and Ear since 2013. His clinical research is focused on corneal imaging—particularly in vivo confocal microscopy—in ocular surface disease and corneal infections. He is also a co-principal investigator in an NIH-funded clinical trial that focuses on reconstruction of the ocular surface using cultivated limbal stem cells. Dr. Kheirkhah’s translational research involves the role of nerve-derived molecules in the maintenance of corneal endothelial cells. Furthermore, in collaboration with scientists at MIT and Northeastern University, he has developed a novel adhesive biomaterial to help repair and regenerate the cornea. To date, his work in the field of cornea and ocular surface disease has led to more than 78 peer-reviewed articles and more than160 local, national, and international presentations.

Dr. Kheirkhah is a member of the editorial board of the Journal of Ophthalmic and Vision Research and is a reviewer for numerous ophthalmology journals. He is also a Moderator/Instructor for several instruction courses at the American Academy of Ophthalmology (AAO) meeting. In addition, he has received an Achievement Award from AAO. ABSTRACT Novel Adhesive Biomaterial for Repair and Regeneration of the Cornea Authors: Ahmad Kheirkhah, Nasim Annabi, Ali Khademhosseini, Reza Dana Currently, repair of corneal stromal defects often requires tissue grafting or cyanoacrylate glue application—both of which have major drawbacks. The aim of our study was to develop an adhesive biomaterial that allows acute repair of corneal defects, while also promoting regeneration of the native cornea. We developed a synthetic hydrogel-based adhesive biomaterial that is solidified by exposure to visible light. Cytocompatibility of the bioadhesive with keratocytes was assessed using 2D and 3D cultures. The bioadhesive was also applied to 50%-deep rabbit corneal defects ex vivo. Transparency, adhesiveness, rigidity, and retention were evaluated using slit lamp biomicroscopy, lap shear test, uniaxial tensile test, and anterior segment optical coherence tomography, respectively. The bioadhesive was also used for 50%-deep rabbit corneal stromal defects in vivo. Biocompatibility and integration of corneal cells into the bioadhesive were then investigated. In vitro tests showed ≥95% cell survival and proliferation/ migration of keratocytes into the bioadhesive. In ex vivo experiments, the bioadhesive was completely transparent with a smooth surface and had a curvature comparable to the normal corneal curvature.

Furthermore, it firmly attached to the corneal defect and had a tensile strength similar to the intact cornea. The bioadhesive remained completely attached to corneas for ≥6 weeks. In vivo experiments demonstrated migration of corneal epithelium over the bioadhesive, growth of keratocytes into the bioadhesive, and minimal inflammation in the cornea surrounding the bioadhesive. Our data show the new light-activated bioadhesive firmly adheres to corneal tissue (for acute repairs) and allows integration of corneal cells (for long-term regeneration).

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30th Biennial Cornea Conference

Alexander V. Ljubimov, PhD, FRSM Professor of Medicine, David Geffen School of Medicine at UCLA; Professor of Biomedical Sciences and Neurosurgery, Director, Regenerative Medicine, Institute Eye Program Cedars-Sinai Medical Center For 14 years, Dr. Alexander has been supervising several laboratories with continuous NIH funding and has been fully funded by the NIH for 20 years.

He has authored more than 100 papers and has served on many NIH and foundation Study Sections nationally and internationally. He is on the editorial boards of 10 scientific journals. He is an Overseas Fellow of Royal Society of Medicine (London) and Gold Fellow of the Association for Research in Vision and Ophthalmology. Dr. Ljubimov is working in the fields of diabetic eye disease and cancer nanotechnology. He pioneered gene therapy for diabetic corneas, where he uncovered epithelial stem cell dysfunction and discovered the angiogenic role of protein kinase CK2. He contributed to the development of polycefins, which are biodegradable, blood-brain-barrier-crossing and cell-targeted polymeric nanodelivery systems for brain and breast cancer treatment. He is combining nanotechnology with gene therapy to normalize aberrant corneal wound healing and is developing corneal epithelial cell generation from normal and diabetic iPSC. ABSTRACT Nanomedicine for Corneal Wound Healing Authors: Alexander V. Ljubimov, Hui Ding, Julia Y. Ljubimova, Mehrnoosh Saghizadeh, Andrei A. Kramerov Gene therapy allows changing gene expression levels to normalize pathological cell behavior. Viral gene therapy vectors provide lasting effects and high efficiency, but may induce immune reactions, inflammation, uncontrolled integration into the host genome, and toxicity to stem cells. This prompted the development of alternative safe gene therapy vehicles. Nanotechnology has produced non-toxic and biodegradable delivery vehicles with efficacy in preclinical models, including acceleration of corneal wound healing. Nanoparticles (NPs) with SMAD7 or BMP7 plasmids or specific siRNAs suppressed stromal haze and scarring after PRK or excimer laser. NPs with Flt23K VEGF intraceptor plasmid prevented corneal neovascularization. These reports emphasized nanomedicine potential to treat corneal wounds. We have developed gene therapy for diabetic corneal epithelial cells. Adenovirus treatment normalized epithelial healing by upregulating c-met and downregulating MMP-10 and cathepsin F in diabetic organ-cultured human corneas, but it was toxic to cultured limbal epithelium requiring lengthy optimization. To circumvent these limitations, we designed non-toxic covalent nanoconjugates (NCs) based on natural-derived polymalic acid, able to change gene expression using antisense technology. Similar NCs significantly inhibited brain and breast tumor growth. NCs were limbal cell-targeted by antibody to transferrin receptor and were internalized through receptor-mediated endocytosis. NC bearing antisense to c-met expression inhibitor miR-409-3p and to cathepsin F increased c-met and decreased cathepsin F expression in diabetic limbal cultures, and NC with MMP-10 antisense inhibited MMP-10. NC modulating c-met and cathepsin F promoted wound healing and increased stem cell marker (ABCG2 and K15) expression in diabetic limbal cultures with no toxicity. NCs did not readily access limbal cells in corneal organ cultures (possibly because of the epithelial layer thickness), showing no effect on wound healing. However, they significantly promoted epithelial healing when used with Lipofectamine. New NCs with a combination of antisense to three c-met miRNAs and two proteinases are currently being tested in diabetic cells and corneas. These data attest to the efficacy of the designed nano gene therapy and its feasibility for diabetic limbal cell normalization.

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Harvard Department of Ophthalmology

Sunil Chauhan, DVM, PhD Associate Professor of Ophthalmology, Harvard Medical School; Associate Scientist, Schepens Eye Research Institute of Mass. Eye and Ear Dr. Sunil Chauhan studies immunomodulation and regenerative medicine in ocular surface inflammatory disorders. He has authored more than 70 original articles in peer-reviewed journals. His laboratory is investigating the function of different immunoregulatory cells, including mesenchymal stem cells and regulatory T cells in the induction of immunity and tolerance in ocular surface inflammatory diseases. Dr. Chauhan’s work has led to many important novel findings, including the involvement of highly pathogenic and autoreactive Th17 cells in the immunopathogenesis of dry eye disease [J Immunol 2009; Blood 2011], and Treg dysfunction in transplantation [J Immunol 2009; J Immunol 2014]. More recently, his laboratory focused on utilizing the immunomodulatory function of mesenchymal stem cells (MSCs) to treat ocular surface disorders [IOVS 2012; IOVS 2014]. The results of preclinical studies are very promising and suggest that the immunomodulatory and regenerative function of MSCs could effectively be used for the treatment of ocular surface inflammation and injuries [Stem Cell Reports 2016; Stem Cells 2017]. Dr. Chauhan’s laboratory has recently demonstrated that MSCs have the capacity to restore corneal transparency by secreting high levels of hepatocyte growth factor (HGF). Interestingly, his results also show that HGF alone can restore corneal transparency, an observation that has translational implications for the development of HGF-based therapy [Molecular Therapy 2017]. Dr. Chauhan continues his work to define the function of immunoregulatory cells, including MSCs, Tregs, and myeloid suppressor cells, with the goal of using their therapeutic potential in ocular inflammation.

ABSTRACT Restoration of Corneal Transparency by Mesenchymal Stem Cells Authors: Sharad Mittal, Masahiro Omoto, Afsaneh Amouzegar, Mingshun Li, Sunil Chauhan Transparency of the cornea is indispensable for optimal vision. Ocular trauma is a leading cause of corneal opacity, leading to millions of cases of blindness annually. Recently, mesenchymal stem cells (MSCs) have gained prominence due to their inflammation-suppressing and tissue-repair functions. They have been used in experimental and clinical settings to treat a variety of tissue injuries and inflammatory diseases. In this study, we demonstrated a hitherto unknown function of MSCs in restoring corneal transparency following ocular injury. Our findings show that MSCs inhibit the expression of opacity inducing α-smooth muscle actin and its inducer transforming growth factor-β in the injured cornea by secreting hepatocyte growth factor (HGF). Furthermore, we show that administration of HGF alone can suppress corneal opacity and inflammation. Given that clinical-grade production of cell-based therapies is cost prohibitive, our findings offer the promise of HGF-based modalities for treating ocular conditions that compromise corneal transparency and vision.

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30th Biennial Cornea Conference

Joaquim Neto Murta, MD, PhD Professor of Ophthalmology, Centro Hospitalar Universitário Coimbra, Faculty Medicine, Coimbra Dr. Joaquim Net Murta specializes in cornea, ocular surface, and cataract and refractive surgery. He has been Professor of Ophthalmology at the University of Coimbra since 2013, and he served as Dean Faculty of Medicine from 2012 to 2015. Additionally, since 2008, he has served as Chairman of the Department Ophthalmology at Centro Hospitalar e Universitário Coimbra. He is also Invited Professor at McGill University in Montreal, Canada, and Mansoura University in Egypt. He holds editorial appointments for several journals, including Revista da Sociedade Portuguesa de Oftalmologia, Revista Portuguesa do Dano Corporal, Revista Brasileira de Oftalmologia, and the Canadian Journal of Ophthalmology.

Additionally, he is a referee for Graefe’s Archives for Clinical and Experimental Ophthalmology, International Ophthalmology, the European Journal of Implant and Refractive Surgery, the Journal of Cataract and Refractive Surgery, Ophthalmologica, the British Journal Ophthalmology, Cell & Tissue Research, and Journal of Refractive Surgery. Dr. Murta has been an active member of several professional organizations.

He was a Member of the Board of the Portuguese Society of Ophthalmology (1989-1990 and 1993- 1994), National Coordinator of the Portuguese Group of Implant-Refractive Surgery (1991-1992), Council Member of the European Society of Cataract and Refractive Surgery (1991-1995), President of the Portuguese Society of Ophthalmology (1997-1998), Member of the Board of the College of Ophthalmology (Ordem Médicos) (19931999), President of the Portuguese College of Ophthalmology (Ordem Médicos) (2000), and Delegate of UEMSSection Ophthalmology (1997-2004) and European Board of Ophthalmology (1997-2003). Currently, he is a Consultant of “Direcção-Geral da Saúde” (2009-present), Member of Research Committee of European Society of Cataract and Refractive Surgeons, Portuguese Representative of International Society of Refractive Surgery of American Academy of Ophthalmology, Member of International College Surgeons, Member of the European Academy of Ophthalmology- Chair XLI, Honorary Member of the Academia Amazonense de Medicina, and Member of European Board Ophthalmology. He has received several awards, including the 2010 Grande Oficial da Ordem do Mérito from the Portuguese Presidency of Republic for his work on ocular transplantation; the 2013 Honorary Member of Academia Amazonense de Medicina, for his work fighting blindness in the Amazon, and the 2013 Achievement Award of American Academy of Ophthalmology.

ABSTRACT Surgical Treatment of Ectatic Corneal Diseases - A Challenge in our Days Authors: Joaquim Neto Murta, Maria João Quadrado, Andreia Rosa and João Gil The incidence and prevalence of ectatic corneal diseases have increased over the last few decades, mainly due to advances in corneal imaging technology and early diagnosis of the disease, as well as the worldwide boom in refractive surgery. The importance of environmental and genetic factors explain the variations across geographical areas. The treatment of ectatic corneal diseases—keratoconus, pellucid marginal degeneration, and iatrogenic corneal ectasia—has changed tremendously over the past two decades.

First intrastromal corneal rings segments (ICRS) were proposed, followed by conventional (C-CXL) and accelerated corneal collagen crosslinking (A-CXL), therapeutic excimer laser treatments. These, in turn, were followed by simultaneous topography-guided photorefractive keratectomy (TG-PRK), followed by corneal cross-linking and other single or combined surgical techniques to treat or prevent the evolution of the disease. Current and emerging surgical options for mild-to-moderate ectatic corneal diseases will be described, based on our experience. This includes the long-term visual and refractive results with different types of intrastromal corneal rings segments (Intacs, Ferrara), based on new keratoconus classifications, pre and post-surgical complications, the efficacy and safety of C-CXL in patients younger than 18 years of age, the outcomes of conventional and accelerated corneal collagen crosslinking protocols, the one-year results of TG-PRK followed by corneal cross-linking, as well as a combination of different surgical techniques.

Also discussed will be the best surgical technique to halt the progression of keratoconus in young patients: CXL or ICRS.

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Harvard Department of Ophthalmology

Saturday, October 14, 2017 Claes H. Dohlman, MD, PhD Professor of Ophthalmology and Chair, Emeritus, Harvard Medical School; Former Chief, Director, Boston Keratoprosthesis, Research and Development, Senior Scientist, Mass. Eye and Ear Dr. Claes H. Dohlman (1922) is an American ophthalmologist of Swedish origins. He was born in Uppsala, Sweden, and attended school in Lund.

He earned his MD in 1950 and then completed ophthalmology training under Professor Sven Larsson. From 1952 to 1954, he completed fellowship training in the United States. Thus, for 18 months he worked under Dr. Jonas Friedenwald at the Johns Hopkins Wilmer Institute, primarily on proteoglycan histochemistry. Eight months were spent at the Retina Foundation in Boston, supervised by Drs. Endre Balazs and Charles Schepens. More ophthalmology training followed back in Sweden. With a continued his interest in cornea biochemistry, Dr. Dohlman earned his doctorate in biochemistry at the Karolinska Institute in Stockholm, where he studied the metabolism of sulfated proteoglycans in the cornea. His preceptors were Professors Lennart Rodén, Harry Boström, Sven Gardell, and Torvard Laurent.

Dr. Dohlman was then promoted to “Docent” at the University of Lund.

In 1958, he received an invitation to come to Boston and work at the then Retina Foundation (now Schepens Eye Research Institute of Mass. Eye and Ear), as well as Mass. Eye and Ear and Harvard Medical School. This was preceded by three months of training in corneal surgery in Lyon, France, under Professor Louis Paufique. After his arrival in Boston, Dr. Dohlman founded the Cornea Service at the Mass. Eye and Ear—the first cornea subspecialty clinic in the world—as well as related fellowship training and clinical research. He also established a laboratory for corneal physiology at the Retina Foundation. These activities grew substantially over the years to become a large referral service for complicated cornea patients, as well as a two-year fellowship training and corresponding research in various aspects of corneal disease.

Dr. Dohlman’s own research during this time changed from biochemistry to corneal physiology and included such problems as corneal edema and corneal nutrition. Also, a number of clinical studies on keratoplasty, corneal edema, herpetic infections, and trauma were published, mostly together with fellows.

In 1968, Dr. Dohlman was appointed Assistant Professor of Ophthalmology at Harvard Medical School, and in 1969, he became Associate Professor of Ophthalmology. In 1974, he was promoted to Professor of Ophthalmology and became Chair of the Harvard Department of Ophthalmology and Chief of Ophthalmology and Director of the Howe Laboratory at Mass. Eye and Ear. He stayed in these administrative positions for a total of 15 years. During this time there was less time for personal involvement in research, but the previously recruited clinical colleagues and scientists continued the established research lines.

In 1989, at the age of 67, Dr. Dohlman retired from his administrative positions but continued full-time work with corneal patients, as well as with teaching and research. His interests gradually became more focused on the development of keratoprosthesis surgery and related biology. He and his clinical and laboratory collaborators developed keratoprosthesis designs, surgical techniques, and postoperative treatment that have made this procedure the most widely used of its kind. More than 13,000 Boston Keratoprosthesis (KPro) devices have been implanted worldwide. His bibliography lists more than 360 publications.

Although Dr. Dohlman stopped performing cornea surgery in 2011, and regular patient hours this year (2017), he is still presently (at age 95) pursuing translational research full time.

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30th Biennial Cornea Conference

Peter Laibson, MD Director of the Cornea Service, Emeritus, Wills Eye Hospital After completing his residency at Wills Eye Hospital in 1964 and a corneal fellowship at Mass. Eye and Ear in 1965, Dr. Peter Laibson returned to Wills to join the Cornea Service. As Professor of Ophthalmology at Thomas Jefferson Medical School, he was Director of the Cornea Service from 1973 to 2000 and is currently its Director Emeritus.

Dr. Laibson was the first full-time cornea specialist at Wills. He and his associates have trained more than 200 corneal fellows and have published more than 400 papers and chapters in the ophthalmic literature. He has been on the editorial boards of many ophthalmology journals during his career and has served on numerous committees of the American Academy of Ophthalmology (AAO). Dr. Laibson has been president of the Cornea Society. He has received many lectureships and awards, including the Lifetime Achievement Award of the AAO (1999) and the Castroviejo Medal and Dohlman Award of the Cornea Society. At the VI World Cornea Congress in 2010, he was one of the five international medalists.

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Harvard Department of Ophthalmology

James Aquavella, MD Catherine E Aquavella Distinguished Professor of Ophthalmology, University of Rochester Flaum Eye Institute Dr. James Aquavella was the first fellowship-trained cornea surgeon when he left Harvard to establish a cornea subspecialty practice in Rochester. He founded the cornea research laboratory at the University of Rochester and continues to be a leader in research, education, and surgery.

He was a founder and past president of the Cornea Society, Director of the Eye Bank Association of America, Board member of Tissue Banks International, and served as a consultant to both the U.S. Food and Drug and Health and Human Services Administrations. As past president of the Contact Lens Association of Ophthalmology, he is well known for his research on the development of the therapeutic contact lenses and the collagen shield. His laboratory was one of the first to use specular microscopy to study endothelial cell morphology and the excimer laser for both therapeutic and cosmetic purposes.

While continuing to provide innovative clinical eye care, Dr. Aquavella is an internationally recognized lecturer, textbook author, and researcher with more than 350 peer reviewed publications. His current research interests include the ocular surface tear film, cornea wound healing, and keratoprosthesis.

He has trained more than 100 clinical, research, and postdoctoral fellows.

He earned his AB from Johns Hopkins University and MD from the University of Naples. He completed his internship at Kings County Medical Center and residency at the then Brooklyn Eye and Ear Hospital.

He subsequently completed clinical and research fellowships at Mass. Eye and Ear and the then Retina Foundation.

A recipient of the American Academy of Ophthalmology Honor, Senior Honor, and Lifetime Awards, Dr. Aquavella has long been included in the listings of “Best Physicians in America” and in the Northeast.

ABSTRACT A Cornea Specialty: The Early Days The creation of the Cornea Service at the Mass Eye and Ear—a major accomplishment of Claes H. Dohlman, MD, PhD—was the first step in establishing the ophthalmic subspecialty of cornea disease.

From his recruitment by Charles L. Schepens, MD, to the then Retina Foundation and the clinical appointment at the behest of Edwin Dunphy, MD—the ensuing six decades have resulted in unparalleled success, as Dr. Dohlman is now considered one of the most honored and respected ophthalmologists in the world.

Dr. Dohlman’s early collaboration with Bent Hedbys, MD, and Saiichi Mishima, MD, DSc, established the basic principles of cornea hydration, while the decades of study and research resulted in the creation of the Boston I Keratoprosthesis, now implanted in more than 13,000 eyes worldwide.

Dr. Dohlman has trained more than 200 fellows and postdoctoral candidates, published of hundreds of research studies, and presented an innumerable number of lectures worldwide, which further attest to the accomplishments of this truly unique ophthalmologist.

The impact of his mentoring instilled in me a deep respect for the never-ending quest for truth and self-criticism as we pursue our research.

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30th Biennial Cornea Conference

Jules L. Baum, MD Professor of Ophthalmology, Emeritus, Tufts University School of Medicine, New England Eye Center Dr. Jules Baum is widely known for his groundbreaking work on tear physiology. He earned his MD from Tufts University School of Medicine, followed by his MS in Ophthalmology at New York University (NYU) School of Medicine. He completed his ophthalmology residency training at Bellevue Hospital, which included two years of research at NYU. Dr. Baum went on to complete a cornea fellowship at Mass. Eye and Ear, working under the mentorship of Dr. Claes H. Dohlman. Dr. Baum returned briefly to NYU-Bellevue Hospital Medical Center before joining the faculty at Tufts. His research has focused on tear physiology; antibiotic pharmacokinetics; ocular infections; pseudomonas attachment to focal deposits on soft contact lenses; and the role of hypoxia in subsequent pseudomonas keratitis, ocular biochemistry, corneal wound healing, and dry eye. He has received many awards throughout his career, including the William Warner Hoppin Award (1959) from the New York Academy of Medicine, the Honor Award (1979) and Senior Honor Award (199) from the American Academy of Ophthalmology, the Alcon Research Institute Award (1991), the Castroviejo Corneal Medal (1997), the Thygeson Award and Lecture from Ocular Microbiology Immunology Group (2001), and the ARVO Gold Fellow Award (2010).

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Harvard Department of Ophthalmology

Stephen D. Klyce, PhD Adjunct Professor of Ophthalmology, Icahn School of Medicine at Mount Sinai Dr. Stephen Klyce began his career in cornea research in 1964 as a Research Assistant to Drs. Saichi Mishima and Claes H. Dohlman at the Retina Foundation in Boston. In 1967, he began studies at Yale University, earning his Physiology PhD in 1971. Between 1972 and 1979, he continued his NEI-sponsored corneal physiology research at Stanford University with David Maurice. In 1979, he transferred his NEI RO1 support to the Department of Ophthalmology, at Louisiana State University School (LSU) of Medicine in New Orleans, where he continued his research in corneal physiology and joined the refractive surgery team. There, he participated in the development of epikeratophakia and excimer laser corneal procedures, and developed the color-coded map for corneal topography analysis. At LSU, he was a Professor of Ophthalmology and Anatomy/Cell Biology and an Adjunct Professor of Biomedical Engineering at Tulane University until July 2008.

Currently, he is an Adjunct Professor of Ophthalmology at the Icahn School of Medicine at Mount Sinai in New York City and serves as a consultant to several ophthalmic industries. He has received professional recognition, including the 1990 CLAO Everett Kinsey Lecture, the 1991 International Society of Refractive Surgery Lans Distinguished Lecturer in Refractive Surgery, the 1991 American Academy of Optometry Max Schapero Memorial Lecture, the 1996 American Academy of Ophthalmology Whitney Sampson Lecture, the 2000 American Society of Cataract and Refractive Surgery Innovator Award, the 2003 International Society for Contact Lens Research Ruben Medal, the 2007 ISRS/AAO Casebeer Lecture Award, the 2009 ARVO Gold Fellow Award, the 2010 ISRS/AAO Barraquer Award, and the 2016 Journal of Refractive Surgery Waring Medal for Editorial Excellence. He serves on the editorial boards of several clinical journals and is an active member of several professional societies, including ARVO (Past President), ISER (Past Councilor), and ISCLR (Past President). He has authored more than 500 publications in the areas of corneal physiology, corneal topography, and keratorefractive surgery.

ABSTRACT Fifty Years After My Launch by Claes Henrik Dohlman, MD In 1964, I came to a fork in the road. Taking the one less traveled (apologies to Robert Frost) has made all the difference. Fresh from college with practical experience in medical device production, I joined the Retina Foundation and measured human tear kinetics and corneal permeability with Saiichi Mishima. Later, I conducted studies on corneal swelling pressure with Dr. Dohlman. This incredible experience taught me the importance of asking the right questions and to never let inconvenient scientific methodology block the path to discovery. But it was Dr. Dohlman—50 years ago—who spurred me on to get my license to do research. That was provided by a PhD in Physiology at Yale studying corneal electrophysiology, and in four short years, I went off to Stanford to join my third career mentor in corneal physiology, David Maurice. This period was followed by 27 years at LSU, where Herb Kaufman introduced me to translational research, and I became interested in refractive surgery. But it was the early guidance from Drs. Mishima and Dohlman that led me to locate the permeability barrier in the cornea—to work out details of then unknown ion transport systems that regulate corneal hydration and transparency, to demonstrate the presence and effect of corneal sympathetic nerve fibers on corneal epithelial physiology, to develop numerical models that explained how the corneal transport systems maintain stromal hydration, to demonstrate how contact lens-induced hypoxia causes corneal edema and Sattler’s Veil, to participate in the development of excimer lasers in keratorefractive surgery, and to develop the color-coded map used to improve refractive surgical techniques and to screen corneas for pathology. It’s been a long and wonderful road; so much is owed to my mentors.

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30th Biennial Cornea Conference

Michael A. Lemp, MD Clinical Professor of Ophthalmology, Georgetown University and George Washington University Dr. Lempâ&#x20AC;&#x2DC;s major research interest is dry eye/ocular surface disease. Over the last four decades he has published 235 peer-reviewed scientific articles and six books and served on national, international, and government advisory boards. He organized and chaired the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eye. He is one of four organizers of the follow up international Dry Eye Workshop (DEWS). He was a member of the steering committee of the Meibomian Gland Workshop report (2011 and the clinical research subcommittee of the DEWS II report. His recent work aims to further understand tear film abnormalities in dry eye and to develop new diagnostic and disease management modalities, most notably tear osmolarity.

After earning his BS (1958) and MD (1962) and completing residency training (1965-68) at Georgetown University, he served as a Fellow in Corneal Disease at the Mass. Eye and Ear and Schepens Eye Research Institute of Mass. Eye and Ear (1968-70), where he co-authored the initial papers on qualitative disorders of the tear film. He has served as Professor and Chairman of the Department of Ophthalmology (1983-1992) at Georgetown University, as an officer and board member of many national and international ophthalmic organizations, and a member of the FDA Ophthalmic Devices Advisory Board and the NIH Study Sections and advisory committees. In 2001 he retired from his clinical practice, but he has continued his teaching and clinical research activities. He is currently Clinical Professor of Ophthalmology at Georgetown and George Washington Universities, the founding Editor-in-Chief of The Ocular Surface, and a scientific advisor to pharmaceutical companies. In addition, he is a scientific reviewer for ophthalmic journals and has served as Chief Medical Officer at TearLab Corp. He has been a Visiting Professor at more than 70 universities, delivered nine named lectures, and in 1998, received the Castroviejo Medal.

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Harvard Department of Ophthalmology

Kathryn Colby, MD, PhD Louis Block Professor of Ophthalmology & Visual Science, Chair, Department of Ophthalmology & Visual Science, University of Chicago Medical Center Dr. Kathryn Colby is an internationally renowned ophthalmologist with expertise in managing complex medical and surgical diseases of the cornea and ocular surface. Dr. Colby has a particular interest in Fuchs’ dystrophy, the most common cause for corneal transplantation in the United States, and is currently pioneering novel treatments for this condition. In addition, she has specific expertise in the management of ocular surface tumors, including conjunctival melanoma and squamous cell carcinoma. She was the founding director of the Pediatric Cornea service at the Boston Children’s Hospital and has recently published the first book devoted to pediatric corneal disease.

Dr. Colby is an active researcher whose contributions have enhanced outcomes for patients who require keratoprosthesis (artificial cornea) surgery. She has an ongoing interest in corneal infections and has published extensively in this area. Dr. Colby was instrumental in determining the optimal surgical techniques for placement of the implantable miniature telescope, a vision-rehabilitative device for patients with macular degeneration.

Dr. Colby is a passionate educator who trained hundreds of medical students, ophthalmology residents, and cornea fellows during the two decades she spent at Harvard Medical School prior to joining the University of Chicago to lead the Department of Ophthalmology and Visual Science as Chair. She currently holds the Louis Block Professorship at the University of Chicago. Dr. Colby lectures around the world on numerous corneal and clinical research topics. She has leadership roles in various national and international societies, including the American Academy of Ophthalmology and the Cornea Society. She has received multiple awards, including the Senior Achievement award and two Secretariat awards from the American Academy of Ophthalmology, and a mentorship award from the American Medical Association.

ABSTRACT Fuchs’ endothelial corneal dystrophy (FECD) occurs in up to 4% of patients in the U.S. and was responsible for 36% of the 47,000 corneal transplants performed in 2016. The corneal endothelium becomes dysfunctional in FECD, causing reduced vision and eventually painful blindness. Despite being described more than 100 years ago, the cause of FECD remains unknown. Multiple cellular pathways have been implicated, including mitochondrial functioning, oxidative stress, apoptosis, epithelialmesenchymal transition, and the unfolded protein response. Multiple genetic abnormalities have been discovered to date, although the vast majority of FECD in Caucasians is caused by a nucleotide repeat expansion on chromosome 18. Tremendous surgical advances have been made in the last 20 years.

Currently, the most common treatment for FECD is selective endothelial replacement, with either Descemet stripping endothelial keratoplasty or Descemet membrane endothelial keratoplasty. Recent evidence suggests that the corneal endothelium in FECD patients may be capable of self-renewal following the removal or destruction of the central dysfunctional endothelium. This talk will review our results with Descemet stripping without endothelial replacement for FECD, including recent results using a topical Rho kinase inhibitor to promote endothelial migration.

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30th Biennial Cornea Conference

Deborah Langston, MD Professor of Ophthalmology, Emerita, Harvard Medical School, Mass. Eye and Ear Biography adapted from James Aquavella, MD, AAO – 2016 Dr. Deborah Pavan-Langston was the first woman accepted into Harvard Medical School’s Ophthalmology Residency Training Program and the first female fellow in Dr. Claes H. Dohlman’s Cornea Fellowship program. She is also, thus far, the only person—man or woman—to win all three of the highest awards in the field of Cornea and External Disease: the Castroviejo Medal, the Thygeson Lectureship, and the Dohlman Society Award.

A graduate of Harvard (Radcliffe) College and Cornell Medical School in NYC, she is a member of both Phi Beta Kappa and Alpha Omega Alpha. Trained in virology by two Nobel laureates (JD Watson & John Enders), Dr. Langston was among the first to study the efficacy and toxicity profiles of antivirals in animal models, later translating these findings successfully to humans. Her expertise is sought quite prominently in national and international health policy for the treatment of ophthalmic herpetic disease, including issues of viral latency, diagnosis, public health, and clinical treatment.

Recently retired, Dr. Langston was the former Chair of the FDA Ophthalmic Drug Advisory Committee, and served on numerous NIH committees, as well as the President’s Commission on Bio-terrorism Preparedness at the Centers for Disease Control and Prevention. She has published more than 250 papers and six books.

Her single-authored text, The Manual of Ocular Diagnosis and Therapy, has been published in six editions and in seven languages. She continues to lecture at Mass. Eye and Ear and serves as a Trustee of the hospital between baby-sitting for her grandchildren.

ABSTRACT Claes H. Dohlman – One Woman’s Opinion Nearing the end of my residency as the first woman trained in ophthalmology at Harvard Ophthalmology/ Mass. Eye and Ear, I realized that I would soon be unemployed. Then, by good fortune, I was assigned to speak on herpetic disease at Grand Rounds. In the audience was Claes H. Dohlman. The very next day, he called and asked if I would be interested in a cornea fellowship. I couldn’t believe it. Like Chairman David Cogan—the man who had first accepted me into the residency program—Claes was not just gender neutral, he strongly favored higher education for women at the doctoral level, including his own daughters.

In my 50 years working at Mass. Eye and Ear, I could not help but notice that early on, women physicians did not exist. My search of the archives revealed not only that they were not considered for training, but also that women were not even welcome in the classroom. Faculty who allowed them in were summarily fired.

Chief Henry Willard Williams was particularly adamant about this.

For 10 years after arriving at Mass. Eye and Ear, I kept my head down and tried not to be offensive. Then in 1975, just after the completion of the new and very grand Mass Eye and Ear, two more women were accepted into the residency—and the floodgates opened. Under Claes’ chairmanship, the numbers rose from 14% to 24%. And by 2004, a woman named Joan Miller, was named the Henry Willard Williams, Professor and Chair of the entire department. Today, women figure prominently at every level, in large part thanks to our esteemed Professor and former Chairman Claes Henrik Dohlman.

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Harvard Department of Ophthalmology

Kenneth Kenyon, MD Clinical Professor of Ophthalmology, Tufts University School of Medicine Dr. Ken Kenyon is an experienced and still active surgeon who specializes in cornea, cataract, refractive and trauma surgeries. An honors graduate of Harvard College and the Johns Hopkins University School of Medicine, Dr Kenyon completed his eye pathology fellowship, residency, and chief residency at the Wilmer Ophthalmological institute of Johns Hopkins. Since then, he has practiced and taught in the Greater Boston area and abroad.

Dr. Kenyon has trained more than 150 clinical and research fellows and has published extensively. Dr. Kenyon is on the faculty of Tufts University School of Medicine, Harvard Medical School, and Schepens Eye Research Institute of Mass. Eye and Ear. He regards his time as a fellow, staff member, and director of the Mass. Eye and Ear Cornea Service (1976-1990) as his professional and personal summit and is eternally grateful to Claes H. Dohlman for this life experience beyond expectation.

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30th Biennial Cornea Conference

Gary N. Foulks, MD, FACS Emeritus Professor of Ophthalmology, University of Louisville School of Medicine When Dr. Gary N. Foulks entered Columbia College in New York City, he initially planned to become a research biochemist, but he later decided to pursue medicine and the applications of biochemistry. He earned his AB from Columbia in 1966 and MD at Columbia College of Physicians and Surgeons in 1970. His ophthalmology residency at the Duke University Eye Center began in 1973. At the encouragement of John Reed, MD (a former Dr. Dohlman fellow), he applied to the Cornea Fellowship at Massachusetts Eye and Ear. He also worked with Richard Thoft, MD, and Judy Friend on ocular surface disease research at the Eye Research Institute of the Retina Foundation. After completing the fellowship, he joined the faculty at the Duke University Eye Center, where he spent 20 years as Professor of Ophthalmology and Director of the Cornea Service. In 1995, he became Professor and Chairman of the Department of Ophthalmology at the University of Pittsburgh. And in 2003, he was appointed the Arthur and Virginia Keeney Professor of Ophthalmology at the University of Louisville in Kentucky. As Director of the Cornea Service, he was also active in clinical and basic research and appointed Associate Dean of Clinical Research until his retirement as Emeritus Professor of Ophthalmology in 2011.

One of the greatest lessons Dr. Foulks learned from Claes H Dohlman, MD, was to appreciate collaborative work with other scientists and PhD researchers in the field. For Dr. Foulks, these included Fred Sanfilippo, MD, PhD; Diane VanHorn Hatchell, PhD; and Gordon Klintworth, MD, PhD, at Duke University. The collaborations continued at the University of Louisville with James Jumblatt, PhD; Marcia Jumblatt, PhD; and Douglas Borchman, PhD. These and other collaborations resulted in 143 peer-reviewed publications, 37 book chapters, and 5 books, as well as the highlight of his career—the 2005 Castroviejo Medal. Dr. Foulks’ gratitude to Dr. Dohlman is deep and sincere.

ABSTRACT Appreciation and Admiration for Claes H Dohlman, MD A tremendous appreciation of the career and accomplishments of Claes H Dohlman, MD, was gained as David Lamberts, MD, and myself prepared a commemorative album (at the encouragement of Jules Baum, MD) in recognition of Dr Dohlman’s retirement as Chairman of the Department of Ophthalmology of the Massachusetts Eye and Ear in 1987. Reviewing the pedigree, training, and accomplishments—both scholastic and familial—of our mentor was gratifying. The most rewarding aspect of that endeavor, however, was to appreciate the profound respect and admiration by which Dr. Dohlman is held by the 80 former fellows who submitted congratulatory letters upon his retirement.

47

Harvard Department of Ophthalmology

Eduardo Alfonso, MD Kathleen & Stanley J. Glaser Chair in Ophthalmology, Professor of Ophthalmology,, Director, Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami Born in Cuba, Dr. Eduardo Alfonso grew up in Puerto Rico. He earned a bachelor degree (summa cum laude, Phi Beta Kappa) and medical degree from Yale University. He completed his residency in ophthalmology at the Bascom Palmer Eye Institute. This was followed by fellowships in cornea and ophthalmic pathology and research at Massachusetts Eye and Ear. He joined the Bascom Palmer Eye Institute University of Miami faculty as Assistant Professor and became tenured Professor and the Edward W.D. Norton Distinguished Chair. Since 2007, Dr. Alfonso has served as the Director. He oversees the clinical, educational, and research programs.

Dr. Alfonso has delivered more than 20 named honor lectures, has been an invited lecturer in more than 200 national and international meetings, and has authored more than 300 scientific publications. He has been certified by the American Board of Ophthalmology and voluntarily re-certified. Dr. Alfonso received the American Academy of Ophthalmology’s Honor Award, Senior Achievement Award, the Life Achievement Honor Award, and a Special Recognition Award. He was named CEO of the Year by South Florida Business, and one of the “100 most influential leaders” in South Florida. He was honored with the Florida Society of Ophthalmology leadership award; the ORBIS International Medical Faculty Award; and was inducted into the American Ophthalmological Society, the University of Miami Iron Arrow Society, and the Academia Ophthalmologica Internationale. He has been named one of the “Best Doctors in America” by Woodward and White, “Top Doctors in the United States” by Castle Connolly, and included on the “100 Most Influential Ophthalmologists Power List” by The Ophthalmologist.

Dr. Alfonso has served as Director and President of the Miami Ophthalmological Society, the Bascom Palmer Eye Institute Alumni Association, Sociedad Iberoamericana de Cornea y Cirugia Refractiva, the Cuban Ophthalmological Society, the Ophthalmology Research Foundation, the Ocular Microbiology and Immunology Group, the Association of University Professors of Ophthalmology, and the Pan American Association of Ophthalmology. He serves on the board of the Heed Ophthalmological Foundation, the Society of Heed Fellows, the Cornea Society, the International Council of Ophthalmology, and the Florida Society of Ophthalmology. He was founder of the Cornea and External Disease Academic Society.

ABSTRACT Fungal Keratitis: Past, Present, and Future The prognosis of fungal keratitis is not promising, especially in developing countries where it primarily affects young, active, working-class males. In addition, prolonged medical treatment, subsequent surgical intervention, and future complications all translate into a high burden of fungal keratitis in these populations. The effective diagnosis and treatment of fungal keratitis remains a challenging area in ocular microbiology. Most of the time, the diagnosis is delayed and correct treatment is not instituted, leading to a poor prognosis for visual function. Risk factors, such as trauma, corticosteroid use, and underlying eye or systemic disease remain well-known culprits. A recent increase in the number of cases of fungal keratitis, particularly those associated with contact lens use, has caused alarm among the public and medical communities. The awareness from the clinician, as well as the use of appropriate diagnostic tools, can prevent catastrophic delays in diagnosis. Current treatment advocates the use of powerful antifungal agents, with eye-preserving surgery for non-responding cases. In this review, every effort was made to appraise and concisely present significant advances in the diagnosis and treatment of fungal keratitis in recent years and to introduce new perspectives and ideas that may be helpful in decreasing the burden of fungal keratitis.

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30th Biennial Cornea Conference

Oliver Schein, MD, MPH Burton E. Grossman Professor of Ophthalmology, Wilmer Eye Institute, Johns Hopkins School of Medicine Dr. Oliver Schein completed his MD in 1981, followed by a residency in internal medicine and an MPHâ&#x20AC;&#x201D;all at Johns Hopkins University. He was sequentially a resident in ophthalmology, corneal fellow, Chief Resident, and then faculty member at Harvard Ophthalmology/Mass. Eye and Ear from 1984 to 1990. He has been at the Wilmer Eye Institute since 1990, where he is the Grossman Professor of Ophthalmology and Vice Chair for Quality and Safety. His research career has been devoted to applying epidemiology to the study of eye diseases and their treatments, and the evaluation of outcomes and risks of ophthalmic devices and surgeries. At Wilmer, in addition to a busy surgical practice, he devotes his energies to optimizing both safety and value in clinical care.

ABSTRACT Claes H. Dohlman: An Enduring Role Model on an Evolving Career Dr. Claes H. Dohlman had a profound effect at a very early stage of my training. I now have more than a 30-year perspective on the variety of ways in which he has been an ongoing role model. I have enjoyed a varied career, with research ranging from large, population-based studies and clinical trials to collaboration on corneal tissue substitutes. This presentation will provide a brief update on my own professional evolution and how the influence of Dr. Dohlman has been there at every step.

49

Harvard Department of Ophthalmology

Deborah Jacobs, MD Associate Professor of Ophthalmology (part-time), Harvard Medical School, Mass. Eye and Ear; Medical Director, Boston Foundation for Sight Dr. Deborah S. Jacobs is an ophthalmologist who specializes in cornea and ocular surface disease. Dr. Jacobs earned an undergraduate AB degree in Biology (summa cum laude) from Harvard and Radcliffe Colleges in 1981 with a thesis on retinal ultra-structure. She was awarded a Rhodes Scholarship to Oxford University, where she earned an MSc in Physiological Sciences in 1983 with a thesis on the development of the visual system.

She attended Harvard Medical School, earning an MD in 1987. Dr. Jacobs trained as an ophthalmology resident from 1988 to 1991, and then as a fellow in Cornea and External Diseases at Mass. Eye and Ear from 1991 to 1992. From 1992 to 2006, she was Director of the Cornea Service at Beth Israel Deaconess Medical Center in Boston. Since 2006, Dr. Jacobs has served as the Medical Director at BostonSight, in Needham, MA. In Dr. Jacobs’ clinical practice at BostonSight, she is devoted to the care of patients with diseases of the cornea and ocular surface and serves as cornea consultant to ophthalmologists and optometrists in the therapeutic contact lens practice. As part of Mass. Eye and Ear’s Cornea Service, she is a consultant in therapeutic contact lens and PROSE treatment. Dr. Jacobs has held leadership roles at Harvard Medical School, the American Academy of Ophthalmology, and the Contact Lens Association of Ophthalmologists. In addition to clinical research on the role of therapeutic lens in the management of cornea and ocular surface disease, Dr. Jacobs has special interests in medical student education, technology assessment, and evidence-based medicine.

ABSTRACT Top-10 Things I Learned from Claes H. Dohlman The years from 1988 to 1992—when I was a resident and fellow—were years of transition at Massachusetts Eye and Ear. In 1989, Frederick Jakobiec succeeded Claes H. Dohlman as Chair of the Department of Ophthalmology. Dr. Dohlman increased his time on the Cornea Service and gave full attention to the KPro project. During that same period, there was a succession of Chiefs on the Cornea Service. Members of Mass. Eye and Ear’s Cornea service during that time included: Claes H. Dohlman, Deborah Langston, Ken Kenyon, C. Stephen Foster, Michael Raizman, Arthur Boruchoff, Ann Bajart, Roger Steinert, Michael Wagoner, and Anthony Adamis, with Eleanor Mobilia and Beth Gould providing contact lens and refraction services. Suffice it to say that an astute trainee could learn a lot! I’m happy to share the Top-10 things I learned from Claes H. Dohlman during my residency and fellowship, and over the 25 years since.

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30th Biennial Cornea Conference

Roberto Pineda II, MD Associate Professor of Ophthalmology, Harvard Medical School; Director, Keratorefractive Surgery Service, Assistant Scientist, Mass. Eye and Ear A native of Minneapolis, Dr. Pineda completed his medical degree at the University of Minnesota Medical School. He was selected to attend the Howard Hughes NIH-HHMI Research Scholars Program at the Cloisters in Bethesda, Maryland, where he worked with retinoblastoma cell lines at the National Eye Institute. He went on to complete residency training at Harvard Ophthalmology, where he served as Chief Resident and Director of the Trauma Service for an additional year. As a Heed Fellow recipient, he completed his Cornea and Refractive Surgery Fellowship at Massachusetts Eye and Ear. Currently the Director of Refractive Surgery at Massachusetts Eye and Ear, and former Division Chief of Ophthalmology at the Brigham and Womenâ&#x20AC;&#x2122;s Hospital, Dr. Pineda has published more than 60 peerreviewed papers and co-authored 5 books, including the award-winning Massachusetts Eye and Ear Infirmary Illustrated Manual of Ophthalmology and the recent second-edition of the Essentials of Cataract Surgery. He has received the American Academy of Ophthalmology (AAO) Senior Achievement Award and has served on the AAO Practicing Ophthalmology Committee in Cataract, the AAO Ethics Committee, and the Preferred Practice Patterns Adult Cataract. He is Co-director of the Harvard Medical School Intensive Cataract Surgical Training Course, now in its 13th year.

He regularly participates in the Flying Eye Hospital Programs run by ORBIS International since 1998. He has been involved in numerous other international programs, as well as clinical research with the International Boston keratoprosthesis program in Ethiopia and Sudan. He is currently working on the development of a new optical imaging device to evaluate the cornea and lens.

ABSTRACT Technology Advances in Presbyopia Correction Presbyopia is considered one of the last frontiers in refractive surgery. The loss of accommodation as we enter middle age is a wake-up call for many about our eyeâ&#x20AC;&#x2122;s decline is physiologic capability. Many approaches to presbyopia have been tried with failure or only limited success in a small group of patients.

Approaches have included scleral structural modification and optical solutions, such as monovision; refractive and diffractive multifocality; and extended depth of focus through the use of contact lenses, cornea laser vision correction, refractive lens exchange, and cornea inlays. Most recently, pharmaceutical options have been tried and are in clinical trials. This talk will review the most recent technology advances to tackle the problem of presbyopia, as well as examine potential future therapies.

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Harvard Department of Ophthalmology

Ilene Gipson, PhD Professor of Ophthalmology, Harvard Medical School; Ocular Surface Scholar and Senior Scientist, Schepens Eye Research Institute of Mass. Eye and Ear Dr. Ilene K. Gipson is a cell biologist and a leading researcher in ocular surface biology. She has been involved in studies of the surface of the eye since l974. Her laboratory contributed to knowledge of corneal epithelial wound healing and anchorage. More recently, her work characterized the secreted and membrane-associated mucins expressed on the ocular surface, and defined their specific function in barrier function and protection from infection and their alterations in drying and cicatrizing eye disease.

Dr. Gipson has authored more than 150 publications and received numerous awards from the academic community, including a Research Career Development Award from the National Eye Institute, an Alcon Award, the MERIT Award from the National Eye Institute, an Honorary Doctoral Degree from her alma mater, Drury College, a Research to Prevent Blindness Senior Investigator Award, the Friedenwald Award from the Association for Vision and Ophthalmology, and the Balazs Award from the International Society of Eye Research. In addition to research activities, she has taught medical students at Harvard Medical School and trained postdoctoral research fellows. Dr. Gipson served as head of the Cornea Ocular Surface Group at the Schepens Eye Research Institute of Mass. Eye and Ear from 1985 to 1997. She has been actively involved in national and international societies for vision and eye research, holding elected offices in the Association for Research in Vision and Ophthalmology, as well as the International Society for Eye Research.

ABSTRACT Claes H. Dohlmanâ&#x20AC;&#x2122;s Legacy in Corneal Research Much has been said and written of the legacy of Claes H. Dohlman, who created the first organized cornea subspecialty and created the first formal cornea fellowship program in this country. Less well known and discussed is his legacy of research that provided fundamental knowledge about corneal physiology and biology that laid the foundation for current understanding of dry eye, corneal ulceration and wound healing, corneal transplantation, and keratoprosthesis.

Dr. Dohlmanâ&#x20AC;&#x2122;s early histochemical and biochemical research in the late 1950s characterized the composition of various matrix component of the cornea. In the 1960s, he and colleagues published physiologic studies determining factors involved in swelling of the cornea, and they investigated tear film evaporation rates, along with the role of collagenase in corneal ulceration. Many of these studies are cited to this day and represent the foundation of current studies in the field.

Not only was Dr. Dohlman exceptionally active in his own research effort, but his inquisitiveness and thorough knowledge of the cornea field also helped others researchers to find fruitful, relevant avenues of research. The corneal research group at the Retina Foundation (subsequently, Eye Research Institute of the Retina Foundation, and more recently, Schepens Eye Research Institute of Mass. Eye and Ear) was a direct outcome of his insight and leadership and is thus another major component of his legacy.

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30th Biennial Cornea Conference

James Chodosh, MD, MPH David Cogan Professor of Ophthalmology in the field of Cornea and External Disease, Harvard Medical School; Associate Director, Cornea and Refractive Surgery Service, Senior Scientist, Mass. Eye and Ear Dr. James Chodosh is the David Glendenning Cogan Professor of Ophthalmology in the field of Cornea and External Disease at Harvard Medical School. He serves as the Associate Director of the Cornea and Refractive Surgery Service, Director of Fellowship Training and Education in Cornea and External Diseases and Surgery, Director of the Ocular Burn Service, and Director of Boston Keratoprosthesis Clinical Programs at Mass Eye and Ear. He has mentored 50 clinical cornea fellows, half of whom now hold academic positions. He is a clinician scientist who is internationally known and respected as a leader for his research and clinical advances in the treatment of severe corneal infections, chemical ocular burns, StevensJohnson syndrome, and keratoprosthesis implantation in end-stage corneal diseases. Dr. Chodosh attended medical school at Baylor College of Medicine, where he also completed his residency training in ophthalmology. He served his fellowship in corneal and external diseases and surgery at the Bascom Palmer Eye Institute, University of Miami, and earned his masterâ&#x20AC;&#x2122;s in public health from the University of Oklahoma. He has authored more than 230 articles and book chapters, and is a four-time award recipient from Research to Prevent Blindness. Dr. Chodosh has performed and taught keratoprosthesis implantation surgery in Brazil, Chile, India, Israel, Italy, the United Kingdom, and Vietnam. He is working on a project to develop a $50 keratoprosthesis for use in less-privileged nations. He is committed to caring for patients with the most severe corneal disorders and seemingly hopeless cases of corneal blindness, as well as training the next generation of cornea specialists. and fellowship, and over the 25 years since.

53

Harvard Department of Ophthalmology

Shahzad Mian, MD Professor in Ophthalmology and Visual Sciences, Associate Chair, Education, Kellogg Eye Center, University of Michigan Dr. Shahzad Mian is Professor in Ophthalmology and Visual Sciences at the University of Michigan/Kellogg Eye Center and holds the Terry J. Bergstrom Collegiate Professorship for Resident Education. His practice is focused in cornea and refractive surgery, and his research interests include corneal transplantation techniques, ocular graft versus host disease, and eye banking. He has been Director of the residency training program since 2004 and serves as the Associate Chair for Education. He completed the Michigan Education Scholarâ&#x20AC;&#x2122;s Program in 2013. In 2003 and 2012, he received the Bergstrom Faculty Teaching Award presented by ophthalmology residents. He serves as the Vice Chair of the Residency Review Committee for Ophthalmology, as the Co-chair of the Accreditation Board for the Eye Bank Association of America, as the Senior Medical Director of Eversight Michigan Eye bank, and is the President of the Program Directorâ&#x20AC;&#x2122;s Council of the Association of University Professor in Ophthalmology.

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30th Biennial Cornea Conference

Mark Rosenblatt, MD, PhD Professor and Head, Department of Ophthalmology & Visual Sciences, Illinois Lions/Charles I. Young Chair in Ocular Research, Director, Corneal Regenerative Medicine Laboratory, Associate Director of Medical Science Training Program, Illinois Eye and Ear Infirmary, University of Illinois at Chicago College of Medicine Dr. Mark Rosenblatt is a corneal surgeon and physician scientist, with a National Institutes of Health-sponsored research program that explores mechanisms to regenerate corneal nerves following corneal injury.

Prior to joining the UIC faculty in October, 2014, Dr. Rosenblatt was Director of the Margaret M. Dyson Vision Research Institute and Vice Chair of Ophthalmology at Weill Cornell Medical College. Dr. Rosenblatt has made significant contributions to our understanding of corneal nerve regeneration, as well as the establishment of innovative models for investigating corneal nerves. At Weill Cornell Medical College, Dr. Rosenblatt expanded his research program to not only include regenerative issues related to corneal nerves, but also to regeneration of the ocular surface. He is currently exploring novel ways to promote trans-differentiation of skin stem cells into corneal epithelial stem cells for the treatment of corneal epithelial stem cell deficiency. In support of these regenerative medicine projects, Dr. Rosenblatt has received funding from the National Institutes of Health, the New York Stem Cell Initiative, the Tri-Institutional Institute for Stem Cell Biology, and SBIR funding from the Department of Defense and the National Science Foundation. Dr. Rosenblatt has published numerous book chapters and papers in peer-reviewed journals, and has presented at national and international academic meetings.

Dr. Rosenblatt’s clinical practice focuses on laser vision correction surgery and the treatment of cataracts and corneal disease. He is board certified in ophthalmology.

ABSTRACT Corneal VEGF Signaling: Angiogenic vs. Neurogenic Functions The identification of the “neurovascular bundle” was identified as an anatomical feature centuries ago.

A number of common signaling pathways have been implicated in modulating both angiogenesis and neurogenesis. The healthy cornea is unique in that it is both avascular and highly innervated. Corneal avascularity and “angiogenic privilege” is a product of the elaboration of a number of anti-angiogenic molecules, with the expression of soluble VEGF receptors (sFlt-1) serving as a key regulator of neovascularization. The upregulation of VEGF in response to corneal pathology overwhelms the inhibitory factors and leads to robust neovascularization. Intact corneal innervation is critical for ocular health, and our laboratory has implicated VEGF as a potent stimulator of nerve repair following corneal injury.

The cornea serves as an excellent model system for identifying critical differences in VEGF signaling in endothelial cells and nerves. We have identified important differences in ligand type (VEGF-A vs VEGF-B) and receptor types (VEGFR-1 vs VEGFR-2 vs NP-1) in mediating VEGF responses in vascular and neural cell types.

55

Harvard Department of Ophthalmology

Victor Perez, MD Professor of Ophthalmology, Director, Ocular Surface Program, Duke University School of Medicine Dr. Victor L. Perez earned his bachelor’s degree in biology from Washington University in St. Louis and medical doctorate from the University of Puerto Rico School of Medicine in San Juan, Puerto Rico. He completed his residency training at Harvard Ophthalmology. Dr. Perez then completed Cornea and External Diseases (Dr. Dimitri Azar) and Uveitis fellowships (Dr. Stephen Foster) at Mass. Eye and Ear. In addition to this, he also trained as an Immunologist Scientist Research Fellow in Ocular Immunology at Schepens Eye Research Institute of Mass. Eye and Ear (Dr. JW Streilein) and a completed a Research Fellowship in Immunology at the Department of Pathology, Brigham & Women’s Hospital in Boston (Dr. Abul K Abbas).

Dr. Perez is a board-certified ophthalmologist by the American Board of Ophthalmology. He was Professor of Ophthalmology and the Director of the Ocular Surface Center and Professor of Ophthalmology at the Bascom Palmer Eye Institute. He was recently named the Stephen and Frances Foster Endowed Chair, Professor of Ophthalmology and Director of the Duke Center of Ocular Immunology at Duke University. His areas of expertise include dry eye, ocular surface inflammation and immunity, and high-risk corneal transplantation with ocular surface reconstruction and uveitis. Dr. Perez is also an NIH-funded investigator and directs the Laboratory of Ocular Immunology, Transplantation and Ocular Surface Regeneration, which is dedicated to developing novel translational concepts with the goal of improving the treatment of ocular surface diseases.

ABSTRACT Lessons I Learned From Dr. Dohlman: The Vitreous is the Enemy of the Keratoprosthesis Authors: Victor L. Perez, Allister Gibbons, Ella H. Leung, Audina M. Berrocal, Thomas A. Albini, Jean Marie Parel, Guillermo Amescua, Eduardo C. Alfonso, and Tayyeba K. Ali The main goal of this work was to determine the impact of total pars plana vitrectomies (PPV), with peripheral shaving of the vitreous base, on the rates of postoperative complications in patients with aphakic, snap-on Type I Boston keratoprostheses (KPros). We demonstrate that the rate of total postoperative complications was lower in the total pars plana vitrectomy group (P=0.018, log-rank test).

In particular, eyes that underwent total PPVs had lower rates of retroprosthetic membranes requiring intervention (P=0.049) and less vision loss attributed to glaucoma progression (P=0.046). There was also a trend for fewer corneal melts (P=0.060) and less sight-threatening complications P=0.051) in the total vitrectomy group. Interestingly, there was no difference in the rates of KPro extrusion (P=0.41), endophthalmitis or vitritis (P=0.15), retinal detachments (P=0.76), cystoid macular edema (P=0.83), or timing of complications between the two groups. With a mean follow-up of 49 ± 22 months, the mean preoperative and postoperative visual acuities were similar between the two groups (P=0.97). In conclusion, eyes that underwent total pars plana vitrectomies during implantation of aphakic, snap-on, Type I Boston KPro had less postoperative complications than eyes with partial or anterior vitrectomies during the average 4 years of follow-up.

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30th Biennial Cornea Conference

Mona Harissi-Dagher, MD, FRCSC, DABO Associate Clinical Professor of Ophthalmology; Chief of Cornea Service, Director of Cornea Fellowship, Director of Ophthalmology Research, Centre Hospitalier Université de Montréal Dr. Mona Dagher completed her undergraduate degree at McGill University, where she received the Hilda Diana Oakley scholarship for ranking topfemale student. She then completed her medical degree and specialization in ophthalmology at the Université de Montréal, graduating on the dean’s honor list. Dr. Dagher then pursued postdoctoral fellowship training in cornea, external eye diseases, and refractive surgery at Mass. Eye and Ear. Dr. Dagher has brought her expertise on keratoprosthesis surgery to Canada after conducting considerable research and clinical work at Harvard Medical School, under the mentorship of Dr. Claes H. Dohlman.

Dr. Dagher is currently an Associate Professor of Ophthalmology at the Université de Montréal, Head of the Cornea Service and Director of the Cornea Fellowship. She is on staff at Hôpital Notre Dame where she teaches medical students, ophthalmology residents, and cornea fellows. She is Director of Ophthalmology Research at Centre Hospitalier de l’université de Montréal, and trains MSc and PhD students. After restoring sight to several Canadian patients through implantation of the Boson keratoprosthesis (KPro), Dr. Dagher was chosen “Personality of the Week” by La Presse newspaper and Radio-Canada. Throughout her academic training, Dr. Dagher was consistently on the honors list and received several scholarships. More recently, she has received research grants from the Fonds de recherche de l’Université de Montréal, the E. A. Baker Foundation for the Prevention of Blindness, the Fondation des maladies de l’œil and the Fondation du Centre Hospitalier de l’Université de Montréal to perform pioneering work in artificial corneal transplantation.

Dr. Dagher has completed extensive research projects in the field of ophthalmology and more particularly, on the Boston KPro and refractive surgery. Dr. Dagher has published numerous articles, book chapters on topics related to cornea and external eye diseases. She serves as a reviewer for ophthalmology journals and has been a guest lecturer at ophthalmology conferences globally.

ABSTRACT Boston Keratoprosthesis: Fresh versus Frozen Corneal Donor Carriers Authors: Adam K Muzychuk, Marie-Claude Robert, Mona Harissi-Dagher The purpose of this study is to examine the comparative long-term safety and efficacy of frozen versus fresh corneal graft carriers of the Boston Keratoprosthesis Type-1 (B-KPro). We conducted a singlecenter, non-blinded, randomized controlled trial comparing Boston KPro surgery with fresh or frozen corneal graft carriers. All patients (37 eyes of 37 patients) who underwent the B-KPro procedure between October 2008 and October 2009 using an allograft carrier were prospectively followed for 24 months postoperatively. Of these, 26 patients participated in an extension study to postoperative 60 months.

The primary outcome of the study was device retention both at 24 and 60 months of follow up.

Secondary measures include pre- and postoperative visual acuity and complications. The Fisher exact test and Student’s t-test were performed to compare the outcome variables. At 24 months of follow up, device retention was 100% (n=37). Of the patients followed to 60 months, 15 eyes received a Boston KPro using a fresh corneal graft carrier and 11 eyes received a frozen carrier. Demographic characteristics were similar between groups. Median pre-operative CDVA in the fresh graft group was CF in the fresh graft group and HM in the frozen group. At 60 months, median CDVA was CF in the fresh graft group, unchanged from baseline, while the median in the frozen group had improved to 20/200. Overall device retention was 96%. There were no significant differences in the rate of complications at 24 and 60 months. Particularly, the occurrence of graft-related complications, such as uveitis or corneal melting, was similar between groups. Five-year data suggest that frozen and fresh corneal donors are equally effective and safe as carriers for the Boston KPro, with no significant differences in device retention or complication rates. The use of frozen tissue may increase the availability of fresh tissue for other procedures, while increasing the accessibility of Boston KPro surgery worldwide.

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Harvard Department of Ophthalmology

Kazuo Tsubota, MD, PhD Professor and Department Chair, Department of Ophthalmology, Keio University School of Medicine Dr. Kazuo Tsubota began his career in eye research 36 years ago during his residency at Keio University School of Medicine in Tokyo (his alma mater). He subsequently completed clinical and research fellowships from 1985 to 1987 in Boston. By around 1995, he became interested in studying age-related eye disorders, such as Fuchsâ&#x20AC;&#x2122; corneal dystrophy, cataract, dry eye, presbyopia, macular degeneration. The Department of Ophthalmology at Keio University School of Medicine has focused its research on aging and regenerative medicine since 2004. An internationally recognized dry eye specialist, Dr. Tsubota has been working on the pathogenesis and treatment of dry eye.

One focus of his research is improving the ocular surface and proper function of vision by assessing functional visual acuity. Other research projects include ocular surface reconstruction, regeneration of the cornea, lacrimal gland, immunology of Sjogrenâ&#x20AC;&#x2122;s syndrome, dry eye, and wound healing of refractive surgery. In addition, Dr. Tsubota serves as President of the Asia Dry Eye Society and the Japanese Dry Eye Society. Dr. Tsubota was the first Japanese recipient of the American Academy of Ophthalmology Honor Award in 1994.

ABSTRACT Science of Tearing Aging decreases tearing. We have hypothesized that an anti-aging approach may recover tearing, which has been proven to be true. Exercise, calorie restriction, a diet rich in antioxidants, and a healthy microbiome can maintain normal tearing in the dry eye model mouse. In contrast, accelerated aging conditions, such as excessive oxidative stress, obesity, or mercury toxicity, have shown a decrease in tearing. Furthermore, we found that tearing excretes heavy metals through the lacrimal gland, but not the salivary gland.

Recently developed DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology enabled us to confirm that the superior salivary nucleus is the functional center for tearing. Although pain and tearing are closely associated, we found that both function differently. At the cellular level, we also found that myoepithelial cells are very important in the acute phase of tear secretion. In addition to the muscarinic receptor stimulation of the lacrimal gland, we recently found the hormonal control of tearing by serotonin.

Finally, the well-known correlation between dry eye and depression prompted us to create an unhappy mouse (stressed model), normal mouse, and happy mouse (enriched environment) to investigate the effect on tearing. The happy mice could maintain tearing through the brain-derived neurotrophic factor (BDNF) axis, but the stressed mouse lost tearing. In the aggregate, these data suggest that dry eye is treatable through anti-aging approaches.

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30th Biennial Cornea Conference

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Harvard Department of Ophthalmology

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30th Biennial Cornea Conference

Poster Abstracts

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Harvard Department of Ophthalmology

01

Inhibition of Corneal Neovascularization by DexamethasoneEluting Contact Lenses in a Rabbit Model Lokendra C. Bengani1,*, Hidenaga Kobashi1, Amy E. Ross1, Hualei Zhai1, Sharad K. Mittal1, Sunil K. Chauhan1, Daniel S. Kohane2, Joseph B. Ciolino1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Department of Anesthesiology, Boston Children’s Hospital, Boston, MA.

1 2

*Email: Lokendra_bengani@meei.harvard.edu PURPOSE: Topical steroids, used clinically for corneal neovascularization inhibition are also used TO control ocular inflammation and reduce corneal scarring. These eye drops are given hourly, with decreasing frequency over a few weeks. Only 1-7% of the instilled dose penetrates the cornea.

In addition, patient adherence is poor. Drug-loaded contact lenses have been proposed as a means of improving drug efficacy and patient compliance. The goal of this study is to evaluate the effects of dexamethasone (Dex)-eluting contact lenses (CL) on experimentally induced corneal neovascularization (CNV) and to compare the efficacy with the current mainstay of treatment, Dex eye drops.

METHODS: CNV was induced by placing 7-0 silk sutures along the superior and inferior cornea of the right eye of NZW rabbits, which were treated with one of the following regimens for one week: no treatment, topical Dex eye drops (0.1 %, 8 hourly drops a day), Dex CL & vehicle CL (no drug) (n = 6). Two masked observers measured the area of CNV from slit lamp photographs taken on day 7. Corneal inflammation was assessed on day 7 by analyzing CD45+ cell frequencies using flow cytometry and corneal neovascularization was evaluated by analyzing VEGF-R2 (KDR) mRNA expression using qRT-PCR in corneal tissue.

RESULTS: The CNV area (mm2) was significantly lesser with Dex-CL (4.0 ± 2.2) than with no treatment (15.0 ± 3.2, p<0.001) or vehicle CL (17.6 ± 2.6, p<0.001), and similar to that from Dex drops (3.4 ± 2.8, p=0.966). The frequencies of CD45+ cells in cornea were significantly lower in the Dex-CL group (5.0 ± 4.5) compared to no treatment (14.8 ± 4.1, p<0.001) or with vehicle CL (10.5 ± 4.0, p=0.023), and were similar to those from Dex drops group (4.8 ± 2.7, p=0.942).

Similar trend were observed in VEGF-R2 (KDR) expression indicating significant reduction in neovascularization by Dex-CL. CONCLUSIONS: Dex-CL effectively inhibited CNV and inflammation, similar to the efficacy of hourly Dex eye drops. CL drug delivery can widen the treatment options for prevention of postkeratoplasty rejection and may serve as a platform for treating other eye diseases.

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30th Biennial Cornea Conference

Multiphoton Intravital Imaging of Plasmacytoid Dendritic Cells in Different Mouse Models of Corneal Inflammation Tomas Blanco1, Arsia Jamali1, Victor G. Sendra1, Maria Lopez1, Hamid-Reza Moein1, Pedram Hamrah1,2

02

Center for Translational Ocular Immunology, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine Boston, MA; 2Cornea Service, New England Eye Center, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 1

PURPOSE: Plasmacytoid dendritic cells (pDCs) play a main role in linking the innate and adaptive immune responses during inflammation. The purpose of this study was to study dynamic pDCs migratory kinetics in vivo through the use of multiphoton intravital microscopy (MP-IVM).

METHODS: Corneal inflammation was induced in a 6-8-week-old DPE-GFP×RAG1-/- transgenic mice (pDC-GFP, n=5/group) by either: placement of 3 intrastromal 11-0 nylon sutures in the paracentral cornea, thermal cautery, or HSV-1 infection (1x106 PFU McKrae). Naïve pDC-GFP mice were used as normal controls. Mice were examined by a MP-IVM system equipped with MaiTai Ti/Sapphire lasers set at 880nm wavelengths. Under anesthesia, mice were imaged at 7 days (suture and HSV-1) or 3 days (cautery) after injury. Distibution, kinetics and velocity of pDCs were evaluated and 4D movies rendered using Bitplane software (IMARIS). The meandering index (MI), an index for directionality (0 random movement, 1 migration in one direction) was additionally calculated. Values from naïve mice (considered as static) and events in inflamed corneas with less than 30 µm of displacement were not included in the MI. Results are shown as means ± SEM. RESULTS: GFP+ pDCs were visualized by MP-IVM of murine corneas. With aid of second harmonic generation pDCs were delineated within the anterior third of the stroma, but not in the posterior stroma or epithelium. pDCs were observed 153±32.55 cells/mm3 in the periphery and 72.84±11.16 in the center (P=0.036) of the naive cornea. In the center, the pDC density increased with respect to the naïve to 144.10±48.49 cells/mm3 (cautery, P=0.081), 150.40± 61.85 (HSV1, P=0.046) and 315.00± 104.50 (suture, P<0.001). The mean velocity of GFP+ pDCs significantly increased from 0.65±0.13 μm/min (naïve) to 3.29 ±1.22 (cautery, P<0.001), 3.67±1.45 (HSV-1, P<0.001), and 4.62±2.03 (suture, P<0.001). The total track length of pDCs in the central cornea significantly increased from 18.62±6.47 µm/hour (naïve) to 127.80±62.22 (cautery, P<0.001), 114.70±54.74 (HSV-1, P<0.001), and 184.4±109.00 (suture, P<0.001). The total displacement length significantly increased from 13.02± 6.47 μm/hour (naïve) to 43.62±23.23 (cautery, P<0.001), 37.11±20.90 (HSV-1, P=0.0023) and (42.19±48.20 (suture, P<0.001). There was no statistical significant difference in meandering index between the inflamed groups 0.36±0.15 (cautery), 0.35±0.18 (HSV-1) and 0.37±0.19 (suture).

CONCLUSIONS: This study demonstrates, for first time, long-term migratory pDC kinetics in different settings of corneal inflammation through high-resolution MP-IVM. These results suggest that pDCs are active components of the corneal immune system that actively respond to inflammation.

FUNDING: NIH-R01-EY022695 (PH), NIH K08-EY020575 (PH), NIH-R21- EY025393

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Harvard Department of Ophthalmology

03

uality of Life and Visual Function in Patients with Boston Q Type I Keratoprosthesis Cristina Bostan1,*, Marwan Elfekhfakh2, Mona Harissi-Dagher1

Department of Ophthalmology, University of Montreal Hospital Center, Montreal, QC, Canada; Department of Family Medicine, University of Montreal, Montreal, QC, Canada

1 2

*Email: bostancristina90@yahoo.ca PURPOSE: To determine patient-reported, vision-related quality of life (VR-QoL) following Boston type I keratoprosthesis (BI-KPro) surgery and its association with postoperative bestcorrected visual acuity (BCVA). METHODS: Descriptive, cross-sectional study. Consecutive consenting patients with BI-KPro were included. The French National Eye Institute Visual Function Questionnaire-25 administered at 51±18 months postoperatively measured VR-QoL. Clinical charts were reviewed for demographics, indications for BI-KPro, baseline, and postoperative (at time of interview) BCVA. For patients operated unilaterally, stratification of VR-QoL scores based on BCVA in the non-operated eye was performed. Multivariate linear regression was carried out, using VRQoL scores as dependent variables, and demographics and postoperative BCVA as covariates.

For patients operated bilaterally, Spearman correlation between VR-QoL scores and BCVA was performed. P<0.05 indicated statistical significance.

RESULTS: Sixty-four patients, aged 59 ± 14 years, 52% male, with a follow-up of 54 ± 19 months, were included. Postoperative BCVA increased from baseline in all operated eyes (p=0.000). In patients with unilateral BI-KPro (n=52), the VR-QoL overall score was 70.7±25.1. Scores on all questionnaire subscales were greater when BCVA in the non-operated eye was >20/200 compared to ≤20/200 (p=0.000). BCVA in the non-operated eye was positively associated with all subscales (p<0.01) independently of age, sex, follow-up duration and postoperative BCVA in the operated eye. In patients with bilateral BI-KPro (n=12) the VR-QoL overall score was 63.0±18.7.

BCVA in the best eye positively correlated with near/distance activities, and social functioning subscales (p<0.05). There was no significant difference between VR-QoL scores of patients operated unilaterally vs. bilaterally.

CONCLUSIONS: We describe VR-QoL more than 4 years after BI-KPro surgery. Compared to data at 1 year previously reported, our results suggest that, as vision progressively deteriorates in the operated eye, patients increasingly rely on their non-operated eye. VR-QoL after bilateral BIKPro is assessed for the first time, and appears comparable to that after unilateral surgery. Larger, prospective, long-term studies, with assessment at baseline, are warranted.

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30th Biennial Cornea Conference

Toxicity of Cosmetic Preservatives on Human Ocular Surface and Adnexal Cells Xiaomin Chen1,2, Yang Liu1, Wendy R. Kam1, David A. Sullivan1

04

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Zhongnan Hospital, Wuhan University, Wuhan, China

1 2

PURPOSE: Cosmetics, such as mascara, eye shadow and eyeliner are used extensively to highlight the eyes and typically contain preservatives to prevent microbial growth. These preservatives include benzalkonium chloride (BAK) and formaldehyde (FA)-releasing compounds, such as quaternium 15, hydantoin, imidazolidinyl urea, diazolidinyl urea and 2-bromo-2nitropropane-l,3-diol. We hypothesize that these preservatives, at concentrations (BAK = 1 mg/ml; FA = 0.74 mg/ml) approved for consumer use, are toxic to human ocular surface and adnexal cells.

Accordingly, we tested the influence of BAK and FA on the morphology, proliferation and signaling ability of immortalized human meibomian gland (iHMGECs), corneal (iHCECs) and conjunctival (iHConjECs) epithelial cells.

METHODS: iHMGECs, iHCECs and iHConjECs were cultured with different concentrations of BAK (5 µg/ml to 0.005 µg/ml) or FA (1mg/ml to 1 µg/ml) under proliferating (keratinocyte serumfree medium, with or without epidermal growth factor and bovine pituitary extract), or differentiating (DMEM/F12 plus 10% fetal bovine serum) conditions for up to 7 days. We used low BAK levels because we found that 0.5 mg/ml and 50 µg/ml BAK killed iHMGECs after 15 minutes exposure.

Experimental procedures included analyses of cell appearance, cell number and Akt signaling in all 3 cell types.

RESULTS: Our results demonstrate that BAK and FA cause dose-dependent changes in the morphology, proliferation and Akt signaling of iHMGECs, iHCECs and iHConjECs. Many of the concentrations tested induced cell atrophy, poor adherence and death, and decreased proliferation after 5 days of exposure. Cellular signaling, as indicated the levels of p-Akt after 15 (FA) or 30 (BAK) minutes of treatment, was also reduced in a dose-dependent fashion in all 3 cell types, irrespective of whether cells had been cultured under proliferating or differentiating conditions.

CONCLUSIONS: Our results support our hypothesis and demonstrate that the cosmetic preservatives, BAK and FA, exert many toxic effects on cells of the ocular surface and adnexa.

FUNDING: This research was supported by the Margaret S. Sinon Scholar in Ocular Surface Research Fund, and the Yong Zhang Research Fund.

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Harvard Department of Ophthalmology

05

Interleukin-7 and -15 Maintain Memory T Helper 17 Cells in Dry Eye Disease Yihe Chen1, Sunil K. Chauhan1, Xuhua Tan1, Reza Dana1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

PURPOSE: Memory T helper 17 (mTh17) cells have been found crucial in mediating the chronicity of various refractory autoimmune disorders, including dry eye disease (DED); however, the underlying mechanisms maintaining mTh17 cells have remained elusive. This study was designed to investigate whether interleukin (IL)-7 and IL-15 maintain mTh17 cells in chronic DED. METHODS: Chronic DED was induced by exposing C57BL/6 mice to desiccating stress using a controlled environment chamber for 14 days and then housing mice in a standard environment for additional 14 days. Expression of IL-7 receptor (IL-7R) and IL-15 receptor (IL-15R) by mTh17 cells (CD4+CD44hiIL-17A+) derived from chronic DED mice was analyzed using flow cytometry. IL-7 and IL-15 mRNA and protein expressions in DED mice were quantified by real-time PCR and ELISA. In addition, draining lymph nodes isolated from chronic DED mice were cultured in the presence of IL-7, IL-15, IL-7 and IL-15, anti-IL-7 antibody (Ab), anti-IL-15 Ab, anti-IL-7 and anti-IL-15 Abs, or control IgG for 72 hours, and then examined for the frequencies of mTh17 cells using flow cytometry. Finally, therapeutic effects of topical neutralization of either IL-7, IL-15 or IL-17 were evaluated in chronic DED. RESULTS: mTh17 cells from chronic DED mice expressed IL-7R and IL-15R. IL-7 and IL-15 expression was significantly higher in chronic DED mice compared to normal mice at both mRNA (2-fold increase, p < 0.05) and protein (37.3±6.2 and 99.2±14.3, vs. 2.0±0.3 and 43.1±4.8, p < 0.05) levels in the conjunctiva. They were constitutively expressed at comparable levels in the DLNs of normal and DED mice. Furthermore, DLNs from chronic DED mice cultured in the presence of IL-7, IL-15, or IL-7 and IL-15 together, displayed higher frequencies of mTh17 cells (0.20±0.03%, 0.18±0.02%, 0.16±0.02%, respectively) compared with those cultured with antiIL-7 Ab (0.057±0.002%), anti-IL-15 Ab (0.055±0.017%), anti-IL-7 and anti-IL-15 Abs together (0.081±0.006%), or control IgG (0.087±0.006%). Topical neutralization of ocular IL-7 or IL-15 effectively reduced DED severity and significantly suppressed frequencies of mTh17 cells in both conjunctiva and DLN, while topical neutralization of IL-17 did not affect mTh17 frequencies in the DLN. CONCLUSIONS: These findings demonstrate that both IL-7 and IL-15 maintain mTh17 cells and could serve as novel therapeutic targets for chronic DED.

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30th Biennial Cornea Conference

Galectin-8-Induced Angiogenesis Is Independent of VEGF-A, but Dependent on Integrins Wei-Sheng Chen1,2,#, Zhiyi Cao1,2, Noorjahan Panjwani1,*

06

New England Eye Center/Department of Ophthalmology, Tufts University, Boston, MA. 2These authors contributed equally.

1

Current affiliation: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD #

*Email: Noorjahan.Panjwani@tufts.edu PURPOSE: Galectin-8 is a tandem-repeat type galectin that possesses two different carbohydrate-recognition domains. Our recent study demonstrated that galectin-8 promotes pathological lymphangiogenesis (the growth of lymphatic vessels) through integrins and podoplanin in the absence of vascular endothelial growth factor receptor-3 (VEGFR-3), the major cell surface receptor that drives lymphangiogenesis. In a separate study, galectin-8 has been shown to promote angiogenesis (the growth of blood vessel). However, it is still elusive whether galectin-8mediated angiogenesis involves VEGF-A/VEGFR-2 signaling axis, the key angiogenic pathway.

METHODS: Human umbilical vein endothelial cell (HUVEC) sprouting assays, corneal micropocket assays, gene knockdown and antibody blocking assays, and galectin-8 knockout mice were used to assess the role and the mechanism of galectin-8-mediated angiogenesis.

RESULTS: In animal models of suture placement and chemical injury in mouse corneas, we showed that inflammatory angiogenesis is markedly reduced in galectin-8 knockout mice. Further, in the corneal micropocket assay, we demonstrated that galectin-8-induced angiogenesis is not affected by VEGF-A blockage. Similarly, in the in vitro 3D sprouting assay by using HUVEC spheroids, galectin-8-induced HUVEC sprouting is not inhibited by a VEGF-A neutralizing antibody. In contrast, blocking antibodies against specific integrins (α5β1, αvβ3 and αvβ5, but not α9β1) markedly attenuated galectin-8-mediated HUVEC sprouting. Using affinity precipitation assays, we demonstrated that galectin-8-associated integrins (α5, αv, β1, β3 and β5) possess complex N-glycans and α2,6-sialyl glycans but not α2,3-sialyl glycans. Moreover, in the in vitro migration assays, we revealed that immobilized galectin-8 promotes HUVEC migration (haptotaxis), whereas soluble galectin-8 has little effect on HUVEC migration (chemotaxis).

CONCLUSIONS: Collectively, our results suggest that galectin-8 functions as an extracellular matrix protein to promote angiogenesis. More importantly, integrins but not VEGF-A/VEGFR-2, play a critical role in galectin-8-mediated angiogenesis.

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Harvard Department of Ophthalmology

07

AAV Vector-Meditated HLA-G Expression Reduces InjuryInduced Corneal Vascularization, Immune Cell Infiltration, and Fibrosis.

Laura Conatser1,2, Brian C. Gilger3, Sara M. Smith3, Jacklyn H. Salmon3, Matthew Hirsch1, 2 Ophthalmology, University of North Carolina, Chapel Hill, NC; 2 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 3College of Veterinary Medicine, North Carolina State University, Raleigh, NC 1

PURPOSE: More than 1.4 million people have corneal vascularization due to genetic and/or environmental factors, leading to diminished visual acuity and often times, blindness. With current treatment options being limited in efficacy and eliciting undesirable effects, we aim to develop an effective therapy to reduce or inhibit injury induced corneal vascularization, that is safer and has long-term results, by investigating an adeno-associated virus (AAV) gene therapy approach designed to exploit the natural immune tolerance mechanism of human leukocyte antigen G (HLA-G).

METHODS: Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLAG-5 (soluble) isoforms were validated in vitro before AAV vector transduction kinetics, using a chimeric AAV capsid, were determined following unilateral corneal wounding in outbred male NZW rabbits. One week after wounding with NaOH burn, all rabbit eyes received a single intrastromal injection of scAAV8G9 vectors encoding optHLA-G1 and optHLA-G5 (optHLA-G Combo), at a 1:1 ratio, or a GFP control. Vascularization and inflammation were monitored by slit-lamp and photography. Fifty-six days after injury, rabbits were euthanized and the eyes were removed, fixed, stained, and evaluated for cornea vasculature, immune cell response, and fibrosis. Serum was analyzed for neutralizing antibodies generated to the AAV capsid and liver, brain, kidney, and heart were collected for vector biodistribution assays.

RESULTS: Intrastromal injections resulted in ~30% corneal coverage and were well tolerated with a complete clearing of the cornea by 24 hrs post-injection. When compared to those that received GFP control vector, rabbits injected with scAAV8G9-optHLA-G Combo had significantly less vascularization (p<.004) and immune cell infiltrate by inhibiting trauma-induced T lymphocyte infiltration of the cornea, some of which were CD8+. Furthermore, immunofluorescence of injured corneas treated with optHLAG Combo vector were negative for alpha-SMA while those treated with the control GFP vector demonstrated a strong signal. Vector genome biodistribution detected AAV vectors only in the cornea; however, approximately 50% of subjects elicited a neutralizing antibody response to the vector capsid following a single administration.

CONCLUSIONS: The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to both, safely and effectively treat corneal vascularization, while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

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30th Biennial Cornea Conference

In vivo Confocal Microscopy Demonstrates Intranasal Neurostimulation-Induced Goblet Cell Alterations in Patients with Dry Eye Disease

08

Gabriela Dieckmann1,2, Paula Kataguiri1,2, Nicholas Pondelis2, Arsia Jamali2, Alessandro Abbouda1,2 Zeina Salem1,2, Manfred Franke3, Michelle Senchyna3, Pedram Hamrah1,2 Cornea Service, New England Eye Center; 2Center for Translational Ocular Immunology, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA; 3Allergan 1

PURPOSE: Goblet cells (GCs), found in the conjunctival epithelium, are specialized mucin secreting cells that contribute to maintenance of tear film stability and ocular surface lubrication.

The Intranasal Tear Neurostimulator (ITN) delivers a small electrical current to sensory neurons of the nasal cavity that stimulate the nasolacrimal reflex and induce tear production. This study compared morphological GC changes before and after use of the ITN by means of laser in vivo corneal confocal microscopy (IVCM).

METHODS: This was a single center, single-arm, study that included 15 dry eye subjects (22 eyes). Laser IVCM images were taken before and after approximately three minutes of ITN application at the inferonasal area of the bulbar conjunctiva. GC were analyzed and measured by ImageJ™ software. Three images from each patient were selected according to their quality and accuracy to be further analyzed by two masked observers. GCs were selected according to the following criteria: 50<area<150 µm2, round or oval shaped, highlighted from the background, well defined borders. All data are shown as mean±SD. Mean data were compared using a paired t-test.

P values <0.05 were considered statistically.

RESULTS: Morphological analysis was performed for a total of 755 GCs pre- and 712 GCs postapplication of the ITN. Mean pre- and post-GC areas were 67.52±40.03µm2 and 58.72±31.16µm2, respectively. The mean change in area, 8.8±8.8 µm, representing a 13.03% reduction following use of the ITN, was statistically significant (p<0.001). Mean pre- and post-stimulation GC perimeters were 48.78±21.84mm and 42.79±19.25µm, respectively. The mean change in perimeter, 5.99±2.59µm, representing a 12.27% reduction following use of the ITN, was statistically significant (p<0.001).

CONCLUSIONS: The ITN significantly reduced GCs area and perimeter within three minutes of stimulation, demonstrating a direct effect on GCs. Laser IVCM is a reliable tool to assess morphological GC changes.

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Harvard Department of Ophthalmology

09

Incomplete Femtosecond Lasik Flaps Secondary to Corneal Epithelial Defect: Case Reports and ex-vivo Studies Hrag Janbatian1,2,3,*, Ali Fadlallah1,2,3, Shaohui Liu2,3,4, Tsai Chu1, Steve Robinson1, Samir Melki1,2,3

Boston Eye Group, Boston, MA; 2Harvard Medical School, Boston; 3Harvard Ophthalmology/ Mass. Eye and Ear; 4Eugene and Marilyn Glick Eye Institute, Indiana University School of Medicine, Indianapolis, IN 1

*Email: hrag.janbatian@bostonlaser.com PURPOSE: To clarify the mechanism by which epithelial defects interfere with LASIK flap creation.

METHODS: Two cases of aborted LASIK procedures were encountered following iatrogenic corneal epithelial defects. The flaps were found to be resistant to lift. To examine the mechanism of flap adherence, 30 fresh porcine cadaver eyes underwent flap creation with a femtosecond laser.

Prior to ablation, the eyes were assigned to 3 groups (10 in each group). Group 1 was assigned as a control group with intact corneal epithelium. In group 2, a central 2.0 mm corneal epithelial defect was created by marking the area with a 2.0 mm trephine. In group 3, a peripheral 2.0mm epithelial defect was created similarly. After the femtosecond laser procedure was performed, the flaps were lifted and the resistance to lift was documented. The corneas were further examined using anterior segment optical coherence tomography (AS-OCT) to image the ablation area under the denuded epithelium.

RESULTS: All 30 porcine eyes underwent uneventful femtosecond ablation. In group 1, no resistance was felt when the flaps were lifted. In group 2, significant resistance was encountered at the edge of epithelial defect during the flap dissection in all 10 eyes, and the flaps could not be lifted in 7 out of 10 eyes. In group 3, similar significant resistance was encountered during the flap lift in all 10 eyes. Six out of 10 flaps could not be lifted. Figure 1 shows an AS-OCT image from a representative cornea from group 2 demonstrating a deeper dissection plane in the area where the overlying epithelium is missing.

CONCLUSIONS: Epithelial defects can interfere with proper LASIK flap creation with the femtosecond laser. This appears to be due to a deeper cutting plane under the area of the epithelial defect compared to the surrounding area with intact epithelium.

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30th Biennial Cornea Conference

Development of a Strategy to Abrogate CD147 in Human Corneal Epithelial Cells with CRISPR/Cas-9 Marissa Feeley1,*, Ashley Woodward1, Jerome Mauris1, Pablo ArgĂźeso1

10

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA.

1

*Email: Feeley_Marissa@meei.harvard.edu PURPOSE: CD147, a glycoprotein that promotes the expression of matrix metalloproteinases, plays a central role in cell proliferation and differentiation. The goal of this study is to develop an efficient strategy to knock-out CD147 in human corneal epithelial cells using CRISPR/Cas-9.

METHODS: We devised two strategies to abrogate CD147 based on mammalian and lentiviral vector systems. Two unique guide RNA sequences were individually cloned into a mammalian vector (PCas-Guide; Origene) and co-transfected with a donor template coding for a fluorescent reporter (RFP) and a blasticidin-resistance gene. The efficiency of transfections was analyzed by immunofluorescence microscopy and immunoblot. The guided RNA for the lentiviral vector system was designed using a software interface (CRISPR Design Tool; MIT).

RESULTS: Cells transfected with the mammalian vector were passaged 10 times to dilute the donor template as episomal form. Thereafter, immunofluorescence microscopy revealed a small population of human corneal epithelial cells expressing detectable levels of RFP. By immunoblot, a 2-fold reduction in CD147 expression was demonstrated with the two CD147-guided RNA vectors compared to scramble control in these cells. Use of the CRISPR Design Tool identified unique 20-mer oligonucleotides targeting exons 1 and 2 of CD147 with minimal offtarget binding. These oligonucleotides were cloned into a LentiCRISPRv2GFP lentivirus and amplified in Stbl3 E. coli.

CONCLUSIONS: Use of a mammalian vector system resulted in partial transfection and abrogation of CD147. A lentivirus approach may be an alternative to improve the efficiency of CRISPR gene editing in human corneal epithelial cells.

FUNDING: NIH R01EY024031

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Harvard Department of Ophthalmology

11

The Impact of Corneal Neovascularization on Visual Acuity and Corneal Sensitivity: Translational Implications Giulio Ferrari1,*, Paolo Rama1

San Raffaele Scientific Institute, Cornea and Ocular Surface Unit, Eye Repair Lab

1

*Email: ferrari.giulio@hsr.it PURPOSE: To quantify the impact of corneal neovascularization (CNV) on visual acuity and corneal sensitivity. Further, to measure the prevalence and etiology of corneal neovascularization in a cohort of patients referred to a third-level cornea clinic.

METHODS: In this retrospective cohort study, 650 patients (803 eyes) were included in the study.

Of these, 497 patients presented with monolateral CNV, and 153 with bilateral CNV. Among these patients, best spectacle corrected visual acuity (BSCVA) and corneal sensitivity were also noted.

Kruskal Wallis and Mann Whitney tests were used to compare: (i) BSCVA and CNV; (ii) BSCVA and sensitivity, respectively. Chi-square test for trend was used to test correlation between corneal sensitivity and CNV. RESULTS: The mean patient age was 54Âą20; M:F=1.13. 28.9% of patients were affected with CNV. Patients affected with CNV had reduced BSCVA. A significant difference was found between BSCVA of patients with involvement of 1 vs 4 (P< 0.001) or 2 vs 4 quadrants (P< 0.01). BSCVA was significantly higher in patients with normal vs. absent corneal sensitivity (P< 0.001). The number of CNV quadrants and corneal sensitivity were inversely related (P< 0.01). The etiology of monolateral CNV was non-infectious keratitis in 47% of cases, infectious keratitis in 41%, and undetermined in 12%. Among infectious keratitis, the vast majority was of viral origin (74%). Finally, we observed that lower visual acuity was associated with deep or extensive (superficial+deep) CNV, when compared with superficial only CNV (P<0.01 and <0.0001, respectively). Moreover, visual acuity was associated with the extent of corneal neovascularization (in quadrants) in patients with superficial CNV (P<0.001), deep CNV (P<0.05), and extensive (superficial+deep) CNV (P<0.01).

CONCLUSIONS: CNV affects one out of three patients referred to a specialist cornea clinic. CNV and its extent are associated with reduced visual acuity and corneal sensitivity.

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30th Biennial Cornea Conference

Corneal Epithelium-derived Thrombospondin-1 Regulates Dendritic Cell Maturation in Dry Eye Disease William Foulsham1, Xuhua Tan1, Yihe Chen1, Afsaneh Amouzegar1, Sunil Chauhan1, Reza Dana1

12

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

PURPOSE: Thrombospondin (TSP)-1 is an important immunoregulatory factor produced by corneal epithelial cells (CECs). The purpose of this study was to investigate TSP-1-mediated regulation of dendritic cell maturation and the therapeutic efficacy of topical TSP-1 in dry eye disease (DED).

METHODS: DED was induced in female C57BL/6 mice using a controlled environmental chamber for 14 days. mRNA and protein expression of TSP-1 in CECs was quantified by real-time PCR and flow cytometry at day 14. Activated bone marrow-derived dendritic cells (DCs) were cocultured with CECs from either na誰ve or DED mice, and the frequencies of MHC-IIhi and CD86+ DCs were evaluated with flow cytometry. These experiments were repeated in the presence of recombinant TSP-1, and also anti-TSP-1 antibody. Finally, DED mice were treated topically with either recombinant TSP-1 or serum albumin. Infiltration and maturation of corneal dendritic cells, expression of inflammatory cytokines, and DED severity were investigated.

RESULTS: mRNA expression of TSP-1 was upregulated in CECs from DED mice compared to na誰ve mice (p<0.001). Although there was no significant difference in the frequencies of TSP-1expressing CECs between na誰ve and DED mice, the mean fluorescence intensity of TSP-1 in CECs from DED mice was significantly increased (p=0.016). The frequencies of MHC-IIhi and CD86+ DCs in the presence of DED corneal epithelium were lower than in the presence of na誰ve corneal epithelium, although the value for CD86+ DCs failed to reach statistical significance (p=0.015 and p=0.117, respectively). Addition of recombinant TSP-1 to the co-culture system resulted in a significant decrease in frequencies of MHC-IIhi and CD86+ DCs (p=0.013 and p=0.008, respectively), while addition of TSP-1 antibody abrogated this suppressive effect (p=0.030 and p=0.015, respectively). Topical administration of recombinant TSP-1 was found to significantly inhibit the maturation and infiltration of corneal DCs, suppress inflammatory cytokine expression at the ocular surface, and reduce the corneal fluorescein staining score in DED mice (p<0.05).

CONCLUSIONS: Our results demonstrate that CEC-derived TSP-1 inhibits DC maturation, and that topical administration of recombinant TSP-1 suppresses ocular inflammation and reduces disease severity in DED mice.

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Harvard Department of Ophthalmology

13

Evaluation of Integrase-Deficient Lentiviral Gene-Transfer Efficiency in Corneal Endothelium Thomas A. Fuchsluger1, Scott Ellis2, Friedrich E. Kruse1, Daniel Thieme1

Department of Ophthalmology, University of Erlangen, Germany; 2Oxford BioMedica, Oxford, United Kingdom 1

PURPOSE: To study the efficacy of integrase-deficient lentiviral vectors in gene transfer to the corneal endothelium: Apoptotic processes in corneal endothelial cells (EC) can result in premature EC degradation. This results in both EC loss during cultivation and also following transplantation.

Gene therapy approaches are explored to prevent EC apoptosis thus extending storage time of donor corneas or to achieve post-operative grafts with higher EC quality. Retroviruses lead to an integration of the DNA of choice into the nucleus, a suboptimal feature in EC modification.

Therefore, we investigated the efficacy of integrase-deficient vector types as potential alternatives for EC transduction.

METHODS: Different types of lentiviral vectors were evaluated regarding their efficiency for shortterm gene transfer. GFP-transducing vectors of EIAV and of HIV origin and their integrase-defective variants were used to transfer genes into human corneal endothelium. Corneal grafts were gained from donors of different age and gender (age 45 ± 24) and stored hypothermic (SightLife, Seattle). Corneas were cut in 4 quarters and each quarter was transduced endothelium-up with the respective vector variants for 24h at 37°C. Fluorescence microscopy was done at 37°C with a Zeiss Spinning Disc confocal microscope endothelium-down. The amount of green fluorescence was quantified via ImageJ. To mimic a gene therapy approach in an eye bank setting, a complete uncut corneal graft was transduced “cornea-in-a-cup-like” with EIAV vector towards endothelium only. Imaging was performed 24 hours after transduction with a non-linear optics imaging system to visualize the corneal endothelium from the epithelial side of the full thickness cornea.

RESULTS: Our studies showed that human corneas can be transduced with all used lentiviral variations, namely EIAV (28 ± 9%), EIAV integrase-defective (38 ± 10%), HIV (17 ± 7.0%), and HIV integrase deficient (24 ± 7.5%). We noticed that integrase-deficient vectors transduced EC more effective than standard integrating vectors (p<0.05). With the standard confocal setting only the endothelium and a small part of the stroma could be investigated. Furthermore, we transduced a complete uncut cornea from the endothelial side and established life cell imaging of all tissue layers of the cornea. We could then visualize healthy and apoptotic, as well as transduced corneal endothelial cells. The transduction efficiency for the corneal endothelium was 75% (± 9%) representing the higher vector to cell relation and we can state now that only endothelium was transduced showing the high specificity of our method.

CONCLUSIONS: Lentiviral viruses are known to be effective gene transfer vectors. With integrasedeficient vectors performing better in the corneal endothelium, a crucial step toward patient treatment is achieved considering regulatory questions. To our knowledge, this study shows, for the first time in the corneal endothelium, that integrase-deficient vectors work as effective as or better than their integrating counterparts. Given confirmation of these data in further experiments in human corneal tissue, we might be closer to translate this concept into an eye bank setting.

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30th Biennial Cornea Conference

Impact of L-Plastin Deficiency in Regulating Innate Responses to Microbiota and Infection Mihaela Gadjeva1, Abirami Kugadas1, Yusha Ru1

14

Brigham and Womenâ&#x20AC;&#x2122;s Hospital, Harvard Medical School, Boston, MA

1

PURPOSE: To evaluate molecular and cellular mechanisms responsible for protection against Pseudomonas aeruginosa keratitis and characterize the influence of the microbiota on antimicrobial immunity.

METHODS: Commensal presence and innate responses were evaluated in L-plastin deficient and sufficient mice. Susceptibility to P. aeruginosa-induced keratitis was characterized.

RESULTS: L-plastin, an actin-bundling protein, regulates microbiota-induced innate and adaptive responses. L-plastin-deficient mice presented with commensal conjunctival overgrowth of Streptococcus ovis consequent to deficient innate responses. Conjunctival IL-6 and IL-17 levels but not IL-1b levels were decreased at baseline in L-plastin KO mice when compared to WT littermates. At a molecular level, L-plastin-deficient macrophages mounted impaired translation of IL-6 transcripts in response to Streptococcus ovis, thereby transmitting reduced neutrophil influx at baseline. Correspondingly, L-plastin deficiency sensitized to P. aeruginosa-induced keratitis.

CONCLUSIONS: Protection against ocular pathogens requires a microbiota-driven maturation of humoral and cellular responses. Even the immune-privileged status of ocular tissues depends on microbes for generating maximal immune resistance to infection. Mechanistically, commensal organisms residing in the conjunctiva regulate antigen presenting cell (APC) motility that is tightly linked to translation of key cytokines, such as IL-6, by the activated APCs. FUNDING: This research was supported by EY022054 (MG).

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Harvard Department of Ophthalmology

15

ISG15 as a Key Player in Corneal Innate Defense Against Pseudomonas Aeruginosa Keratitis Nan Gao1, Fu-shin Yu1

Kresge Eye Institute, Detroit, MI

1

PURPOSE: To elucidate the role of ISG15 in controlling corneal inflammation and innate immunity in B6 mouse model of Pseudomonas aeruginosa keratitis.

METHODS: Cultured primary HCECs were challenged with 100:1 HK PA. Cells or conditioned media were collected at 4-16 h for Western blotting analysis. To determine the role of ISG15 signaling in P. aeruginosa keratitis, the corneas of WT or ISG15 KO mouse corneas were inoculated with 104 P. aeruginosa. Disease progress was monitored by digital photograph, clinical scoring, and MPO measurement for PMN infiltration. The expressions of various pro-inflammatory and innate defense genes were determined by real-time PCR. Cryostat sections of mouse corneas were immunostained with antibodies against F4/80, iNOS, ARG1 and PMN. Macrophages were collected from WT or ISG15 KO mice peritoneal cavity and treated with heat-killed PA for 1 hour and subjected to real-time PCR analysis.

RESULTS: Heat-killed ATCC infection induced the expressions of ISG15 in PHECEs and conditioned media. Knockdown of ISG15 greatly increased severity of keratitis, including markedly increased bacterial burden and PMN infiltration. Knockdown ISG15 induce downregulation AMPs while and upregulate the expression of IL1β. ISG15 deficiency increased infiltration of PMN and macrophages, both type 1 and 2, in infected corneas. In vitro analysis of cultured macrophages revealed that challenging of isolated macrophages with heat-killed PA markedly induced the expression of IL-1β, IL-23, Arg1, CXCL2, iNOS in an ISG15-dependent manner, as well as CD206 and CXCL10 in an ISG15-independent manner.

CONCLUSIONS: ISG15 is an innate immune response gene and its deficiency increases corneal susceptibility to P. aeruginosa and altered the expressions of many cytokines and defense molecules in vivo and in cultured macrophages.

FUNDING: NIH grants R01 EY017960, EY010869

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30th Biennial Cornea Conference

Vasculogenesis after Excimer Laser PTK Daniel J. Gibson1,*, Soojung Seo1 University of Florida, Institute for Wound Research

1

16

*Email: gibsondj@ufl.edu PURPOSE: In monitoring the process of active haze formation in excimer laser injured corneas, two distinct regions of haze initiation and growth were observed. Micrographs have revealed that the peripheral injury haze cells have morphologies in keeping with myofibroblasts, while those in the middle appear to be vascular. These corneas did not have any vessel ingrowth from the limbus. In continuing to test the hypothesis that the central haze is vascular in nature, a panel of targets was assembled to assess the presence and levels of growth factors and endpoints which related with the VEGF family and its byproducts.

METHODS: Wounded corneas were collected using an 8mm punch biopsy. Samples underwent reverse transcriptase reaction with the IScript cDNA Synthesis Kit (Bio-Rad) using gene-specific primers to generate cDNA. Real-time PCR was then performed using the ABI 7300 qPCR system (Applied Biosystems) with SYBR Green qPCR Master Mix (Applied Biosystems). Specific genes from fibrosis and vasculogenesis pathways were assessed including: collagen I, III, V, Îą-SMA, CD31, laminin, VEGF-A, PLGF, VEGF receptor I, VEGF receptor II, ANGI, ANGII, TIE1, CD34, & ALDH-1. Following the qPCR results, VEGF-A protein levels were assessed via ELISA (R&D system) and CD31 findings were affirmed via immunohistochemical staining.

RESULTS: We have begun an analysis of mRNA quantification of several genes in the fibrosis and vasculogenesis related pathways and this has yielded some interesting and promising results. Both fibrosis and vasculogenesis markers were elevated in the cornea quantified using qPCR. ALDH-1 was down regulated which is a marker for haze. Fibrosis related markers, Collagen III and Îą-SMA mRNA levels were increased in injured corneas compared to non-injured corneas. Increased vasculogenesis markers, VEGF-A, CD31, VEGFR2, PLFGF, and laminin were also observed in injured corneas. The levels of CD-31 in a cornea with central haze are in direct proportion to the level of the growth factor responsible for vessel growth (VEGF-A). VEGF ELISA data and CD31 immunostaining affirmed these results.

CONCLUSIONS: Our findings strongly suggest that vasculogenesis plays a key role in central corneal haze formation.

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Harvard Department of Ophthalmology

17

Improving the Practicality and Safeness of Artificial Corneas: Preassembly and Gamma Sterilization of the Boston Keratoprosthesis

Miguel Gonzalez-Andrades1,*, Mohammad M. Islam1, Thibaut Divoux2,3, Michael Haist4,5, Sina Sharifi1, Eleftherios Paschalis1, Andrea Cruzat1, Larisa 1 Gelfand , Franz-Josef Ulm5, James Chodosh1, Francois Delori1, Claes H. Dohlman1 Massachusetts Eye and Ear and Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, United States; 2Centre de Recherche Paul Pascal, Pessac, France; 3MultiScale Material Science for Energy and Environment, UMI, Massachusetts, United States; 4Institute of Concrete Structures and Building Materials, Karlsruhe Institute of Technology, Karlsruhe, Germany; 5Massachusetts Institute of Technology, Civil and Environmental Engineering Department, Massachusetts, United States 1

*Email: miguel_gonzalez@meei.harvard.edu PURPOSE: Presently, the Boston keratoprosthesis (B-KPro) is implanted into a corneal graft by the surgeon in the operating room. The combination is then sutured into the patient’s eye. This routine has led to occasional errors and the logistics are cumbersome and expensive. A simplified method is proposed. The B-KPro can be preassembled into a corneal graft and then transferred to a vial to be sterilized with gamma-radiation (approved method for corneal grafts). The resulting combination can then be stored and transported. For such a procedure to be acceptable, it has to be shown that gamma-radiation will not damage the B-KPro. METHODS: We have evaluated the effect of gamma-radiation on the medical PMMA that is used in the manufacturing of the B-KPro. 15mm-diameter discs of PMMA were submitted to either ethylene oxide sterilization (the presently used process for the B-KPro), or different doses of gamma-radiation (10, 25 and 50 kGy), independently. Cell biocompatibility, mechanical strength and optical quality of the material were evaluated. Moreover, the feasibility of assembling the B-KPro to an allograft and gamma-irradiate afterwards was also evaluated.

RESULTS: There were no differences in cell biocompatibility among the samples, after culturing corneal epithelial cells and fibroblasts in contact with them (p>0.05). The mechanical evaluation by statistical nanoindentation showed no alterations of the PMMA after irradiation. The optical evaluation showed high levels of transparency for the ethylene oxide, 10 kGy, and 25 kGy groups.

The absorbance of ultraviolet was higher for the 25 kGy and 50 kGy groups. Technically, preassembly and irradiation of the B-KPro revealed no problems.

CONCLUSIONS: Sterilization of the B-KPro using gamma-radiation has no detectable influence on the biocompatibility, mechanical or optical properties of the device. Pre-assembling the B-KPro to a donor cornea, followed by sterilization with gamma-radiation—allowing long-term storage and easy shipment—emerges as an efficient and safe procedure.

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30th Biennial Cornea Conference

Descemet’s Membrane Formation in a 3D Culture Model Xiaoqing Guo *, Audrey E. K. Hutcheon , James D. Zieske 1,

1

1

18

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

*Email: Xiaoqing_Guo@meei.harvard.edu PURPOSE: The goal of this project was to examine if our 3-dimensional (3D) stromal matrix provides a good substrate to promote human corneal endothelial (HCEndo) maturation and Descemet’s membrane (DM) formation.

METHODS: Human corneal fibroblasts (HCFs) in 6-well transwell plates were cultured in EMEM + 10% FBS + 0.5mM VitC for 3 weeks. Then, HCEndo cell line (HCEndoCL) was seeded onto the HCF-stromal matrix for up to 2 weeks. To examine for DM formation, the HCEndoCL-HCF cocultures were assayed for the expression of Laminin, a component of DM, and observed by TEM. To examine for maturation, the expression of ALDH1A1, a marker of mature endothelial cells, was assayed.

RESULTS: We observed copious amounts of Laminin that was deposited at the interface of the endothelial cells and the stromal matrix, as well as structures by TEM that corresponded to early stages of DM formation. We also found that in confluent or single HCEndoCL cultured on glass slides, little, if any, ALDH1A1 was present; however, in co-culture tissue ALDH1A1 staining was positive in both the HCEndoCL and HCF cytoplasm.

CONCLUSIONS: 3D matrix is a good substrate for HCEndo maturation. We postulate that HCEndo injected into the human corneal anterior chamber will attach to the host’s posterior stroma and develop into a mature endothelium and secrete new DM components.

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Harvard Department of Ophthalmology

19

Evaluation of Transepithelial Corneal Collagen Crosslinking (TECXL) as a Safe and Effective Treatment to Prevent Progression of Ectatic Conditions of the Cornea Kathryn M. Hatch1, TavĂŠ van Zyl2, Aaron Young3, Jonathan H. Talamo4

Mass. Eye and Ear, Waltham; Harvard Ophthalmology; 2Mass. Eye and Ear/Harvard Ophthalmology; 3Brandeis University; 4Mass. Eye and Ear/Harvard Ophthalmology, Johnson and Johnson Vision 1

PURPOSE: To evaluate the safety and efficacy of a transepithelial technique of collagen crosslinking (TECXL) for the treatment of ectatic conditions of the cornea.

METHODS: We conducted a single center retrospective chart review of patients undergoing TECXL at Talamo Hatch Laser Eye Consultants/Mass. Eye and Ear, Waltham between 6/4/13 and 4/16/16. We used a proprietary riboflavin solution and loading system followed by pulsed UVA light for 30 minutes at 4 mW/cm2. Preoperative and post-operative best-corrected (BCVA), as well as steepest radius of curvature (Kmax) were assessed. Follow-up visits occurred at 3, 6 and 12 months. A continuous cohort beyond 12 months was also assessed. Retreatment rates and adverse events, including visually significant corneal haze and infectious keratitis were also assessed. A sub-analysis of eyes which did not undergo a soft contact lens holiday of greater than 7 days was analyzed.

RESULTS: 320 eyes of 200 patients met inclusion criteria. The average patient age was 31.4 years (SD 12.4 years). Mean pre-operative BCVA was 0.3 LogMAR (SD 0.3), and mean KMax was 58.4 D (SD 10.9 D). The most common indication for TECXL was keratoconus (93.5%) followed by post-LASIK ectasia (3.1%) and pellucid marginal degeneration (1.6%). There were no adverse events including visually significant haze or infectious keratitis. The retreatment rate due to signs of topographic progression was 2.5% (n=8). Among these eyes, none had lost greater than 2 lines of BCVA prior to retreatment.

CONCLUSIONS: TECXL using a proprietary riboflavin and loading system appears to a safe and effective-promising treatment for stabilization of ectatic conditions of the cornea. Retreatments can be considered to increase the effects of CXL.

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30th Biennial Cornea Conference

Prevalence and Risk Factors of Exposure Keratopathy Across Different Intensive Care Units Juliet Hartford1,*, Priya Mathews1, Joaquin deRojas1, Aakriti Garg1, Nailyn Rasool1, Sara Schroder2, Danielle Trief1

20

Department of Ophthalmology Edward S. Harkness Eye Institute, New York, NY; 2Barnard College, New York, NY 1

*Email: jbh2157@cumc.columbia.edu PURPOSE: To determine the prevalence and risk factors of exposure keratopathy (EK) across different ICUs, specifically in Columbia University Medical Center’s (CUMC) Pediatric (PICU), Medical (MICU), and Neurologic (NICU) Intensive Care Units (ICU).

METHODS: In this prospective cohort study, 46 sequential patients were examined daily, with the initial examination occurring within 24 hours of admission, in the PICU (8 patients), MICU (15 patients) and NICU (23 patients). Data on eyelid position, conjunctival and corneal changes, which included presence of conjunctival injection and punctate staining on fluorescein exam, Bell’s and blink reflex, medications, Glasgow Coma Scale (GCS) rating, and ventilation type were collected.

Logistic regression models were estimated using generalized estimating equations with the outcome being the presence of EK as defined by punctate staining seen on fluorescein exam in the inferior 1/3 of the cornea.

RESULTS: The EK rates overall were PICU 25%, MICU 60% and NICU 48%. These differences weren’t significant (p=0.28). Factors with increased odds of EK were lagophthalmos (OR=16.7, p=0.00, 95% CI 7.4-37.5), conjunctival injection (OR=2.8, p=0.02, 95% CI 1.14-6.8), GCS (OR=.84, p=0.00, 95% CI 0.76-0.9), intubation (OR=2.8, p=0.01, 95% CI 1.3-5.9), opioids (OR=2.6, p=0.01, 95% CI 1.2-5.5) and sedation (OR=2.6, p=0.01, 95% CI 1.2-5.5), and didn’t differ across ICUs. ICU differences included admission reason (p=0.00), sepsis (PICU 25%, MICU 60%, NICU 4%, p=0.00), Bell’s reflex (PICU 100%, MICU 73%, NICU 52% p=0.04), and surgery (PICU 37.5%, MICU 0%, NICU 4.4%, p=0.00).

CONCLUSIONS: This was the first study to examine rates of EK across ICUs. PICU patients were more likely to have a Bell’s reflex and the PICU trended towards decreased rates of EK, yet did not reach statistical significance. Across all ICUs lagophthalmos, conjunctival injection, low GCS rating, intubation, and the use of opioid and sedative medications were associated with increased odds of EK.

81

Harvard Department of Ophthalmology

21

Anti-CD80/86 Injection Prolongs the Graft Survival in Murine Corneal Transplantation Osama Ibrahim Hirayama1*, Keiichi Fujimoto2, Takenori Inomata2, Koichiro Uchida3, Tina Shiang4, Akira Murakami2

The University of Tokyo Hospital, Department of Ophthalmology; 2Juntendo University School of Medicine, Department of Ophthalmology; 3Juntendo University School of Medicine, Atopic Disease Center; 4Boston University School of Medicine, Department of Ophthalmology 1

*Email: osamaibrahim82@gmail.com PURPOSE: To assess the immune responses and the effects on graft survival of anti-CD80/86 injection in murine corneal transplantation.

METHODS: Three intrastromal sutures were placed in the recipient graft bed of the high-risk (HR) mice group two weeks before allogeneic transplantation to induce angiogenesis and amplify the risk for graft rejection. Control low-risk (LR) graft recipients did not undergo suture placement, and thus the host bed remained avascular at the time of transplantation. Anti-CD80/86 was administered to transplanted mice by intraperitoneal injection immediately after and 24 and 48 hours post transplantation. (PBS was administered as control.) Graft opacity scores were evaluated for 8 weeks by slit-lamp biomicroscopy. We used a standard opacity-grading (range, 0-5+) scheme to define rejection; corneas with an opacity score of 2+ for two consecutive examinations were considered rejected. Ipsilateral draining lymph nodes (dLNs) were harvested 14 days after transplantation. dLNs were analyzed for CD4+CD25+Foxp3+regulatory T cells (Tregs), CTLA-4, CD11c+ dendritic cells (DC) and CD11b+ macrophage by flow cytometry. The expression level of Foxp3, CTLA-4 in Tregs and MHC class II in CD11c+ DCs were analyzed by mean fluorescence intensity (MFI) using flow cytometery. The study was conducted in compliance with the ARVO statement for the use of animals in Ophthalmic and Visual Research.

RESULTS: Anti-CD80/86 injection prolonged graft survival in both groups, especially in HR grafts (mean survival, LR-PBS; 44.8 days, LR-anti-CD80/86; 56 days, HR-PBS; 24.5 days, HRanti-CD80/86; 49 days, p<0.001). Anti-CD80/86 administration reduced the graft opacity score in HR-anti-CD80/86 compared to HR-PBS at day 56 post-transplantation (LR-PBS; 2.3, LRanti-CD80/86; 1.2, HR-PBS; 5.0, HR-anti-CD80/86; 1.3, p<0.05) Anti-CD80/86 administration increased MFI expression level of CTLA-4 in dLNs of HR-anti-CD80/86 compared to HR-PBS (MFI, LR-PBS: 4100, LR-anti-CD80/86: 4150, HR-PBS: 3462, HR-anti-CD80/86: 4229, p=0.0025).

The frequencies and the MFI levels of foxp3 showed no significant difference after anti-CD80/86 administration. The MFI expression of MHC class II in CD11c+ DCs of HR-anti-CD80/86 was reduced compared to HR-PBS (HR-PBS; 45748 vs. HR-anti-CD80/86; 34566, p<0.05).

CONCLUSIONS: We found that blockade of CD80/86 at the time of transplantation induced longterm allograft survival via CTLA-4 activation and MHC class II deactivation.

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30th Biennial Cornea Conference

Fiber-Optic Pressure Sensor Integrated in the Boston Keratoprosthesis for Glaucoma Screening Pui-Chuen Hui1,*, Néstor Uribe-Patarroyo2, Claes H. Dohlman1, Brett E. Bouma2, Eleftherios Paschalis Ilios1

22

Schepens Eye Research Institute of Mass. Eye and Ear, Boston Keratoprosthesis Laboratory, Harvard Ophthalmology, Boston, MA; 2Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Ophthalmology, Boston, MA, 1

*Email: puichuen_hui@meei.harvard.edu PURPOSE: Elevated intraocular pressure (IOP), a risk factor of glaucoma, is one of the complications of prosthokeratoplasty. Currently, IOP is assessed via the subjective palpation method since conventional tonometry is unsuitable for the rigid corneal implant. In this study we design and evaluate a novel miniaturized fiber-optic pressure sensor (PS) integrated in the Boston keratoprosthesis (B-KPro) device for quantitative IOP measurements.

METHODS: A fiber-optic Fabry-Perot pressure sensor with a small footprint (diameter: 300 um, length: 3 mm) was fitted in the B-KPro’s optical stem. IOP measurements were performed using eye-safe broadband illumination, which was delivered to the PS externally via a bench-top sweptsource optical coherence tomography (OCT) system. The IOP response was assessed by spectral analysis of the PS’s interferometric fringes. Competing interferograms from weaker scatterers that mask the desired spectral information were temporarily removed by blocking the reference arm in the OCT interferometer. The dynamic range and stability of the PS were independently determined by continuous measurements at different atmospheric pressures for 24 months.

RESULTS: A prototype B-KPro device was equipped with the fiber-optic IOP sensor and tested.

IOP measurements were performed by focusing at the strongest reflection in the OCT B-scan, from which we obtained interferometric fringes that corresponded to the PS at atmospheric pressure.

Independent continuous manometry measurement of two PSs demonstrated low pressure drift over 2 years (<0.3 mmHg) within a dynamic range of 50 mmHg. CONCLUSIONS: Integration of a fiber-optic pressure sensor in the B-KPro device is feasible. The low-drift response of the system makes it highly attractive for clinical use in B-KPro patients. The ability to acquire IOP reading using clinically available OCT systems substantially simplifies the use of the system and may help to improve glaucoma management in B-KPro patients.

83

Harvard Department of Ophthalmology

23

Cyclophotocoagulation Controls Intraocular Pressure in Patients with the Boston Keratoprosthesis Type 1 and Glaucoma Samir Jabbour1,*, Marie-Claude Robert1, Mona Harissi-Dagher1

Department of Ophthalmology, HĂ´pital Notre-Dame (CHUM), Montreal, Quebec Canada

1

*Email: samir.jabbour2@gmail.com PURPOSE: To report the outcomes of cyclophotocoagulation (CPC) in controlling intraocular pressure (IOP) and progression of glaucoma in eyes with the Boston keratoprosthesis (B-KPro) type I. METHODS: Retrospective chart review of 18 eyes in 17 patients with the B-KPro who underwent CPC for glaucoma control. All CPC were done after B-KPro implantation. IOP, best-corrected visual acuity (BCVA), and number of glaucoma medications were recorded preoperatively and postoperatively at week 1, months 1-3, months 4-6, and at last visit. All CPC-related complications and the need for other interventions were also recorded.

RESULTS: The mean age of patients at CPC was 78 +/- 13 years with a mean follow-up period of 24 +/- 20.6 months [range 1 â&#x20AC;&#x201C; 73 months]. The most common indication for CPC administration was uncontrolled IOP despite maximal tolerated medical therapy and glaucoma drainage device (n=9), followed by avoidance of intraocular surgery in an eye with poor visual potential (n=4).

Mean pre-operative IOP was 24 +/- 5 mmHg and was reduced to 14 +/- 8 mmHg at week 1 post-operatively (p<0.001), to 15 +/- 4 mmHg at months 1-3 post-operatively (p<0.001), to 15 +/- 5 mmHg at 4-6 months post-operatively (p<0.001) and to 15 +/- 4 mmHg at last follow-up (p<0.001). Number of glaucoma medications (including topical drops and acetazolamide per-os) was 3.9 +/- 0.8 pre-operatively compared to 3.2 +/- 1.3 medications at 6 months post-operatively (p~0.3) and 3.2 +/- 1.8 at last follow-up (p~0.5). Pre-operative BCVA was 1.2 +/- 1. logMAR pre-operatively and was slightly reduced to 1.4 +/- 1.2 logMAR at 3 months post-operatively (p~0.7) and to 1.3 +/- 1.2 logMAR at last follow-up (p~0.8). Overall 7/18 patients had evidence of glaucoma progression after CPC treatment. One patient had a retrobulbar hemorrhage secondary to retrobulbar anesthesia. Two patients experienced hypotony during the first month following CPC and one patient had endophthalmitis in the week following CPC. Three patients required a second CPC for further control of the IOP. CONCLUSIONS: CPC contributes to IOP control in eyes with refractory glaucoma after B-KPro type 1 implantation but did not lead to a significant reduction in the number of glaucoma medications administered. Glaucoma can progress despite. However, other severe complications were not common.

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30th Biennial Cornea Conference

Plasmacytoid Dendritic Cell-based Therapy for Corneal Nerve Regeneration Arsia Jamali1, Maria J. Lopez1, Victor G. Sendra1, Deshea L. Harris1, Nicholas J. Pondelis1, Pedram Hamrah1,2

24

Center for Translational Ocular Immunology, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA; 2Cornea Service, New England Eye Center, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 1

PURPOSE: We have recently shown that plasmacytoid dendritic cells (pDCs) exert neurotrophic properties in the cornea. The aim of this study is to evaluate the therapeutic efficacy of local adoptive transfer of pDCs in corneal nerve regeneration.

METHODS: Corneas of 6-8 week-old male wildtype (WT) C57BL/6 mice underwent deep stromal trephination to sever corneal nerves. Splenic GFP+ pDCs from DPE-GFP×RAG1-/- mice and WT CD11b+ myeloid cells were isolated. After trephination, 104 pDCs, CD11b+ cells, or PBS control were locally applied onto the corneas using tissue glue. On day 3, corneas underwent flow cytometry to assess protein expression of NGF. On day 14, corneas were stained for βIII-tubulin (pan-neuronal marker), CD45 (pan-leukocyte marker), and MHC-II (maturation marker). Total length of corneal nerves was quantified via NeuronJ and densities of MHC-II+ cells were measured by ImageJ. ANOVA with LSD post hoc test was used to assess statistical significance. p<0.05 was considered significant.

RESULTS: Confocal microscopy confirmed successful transfer of GFP+ pDCs to both central (331.5±42.7 cells/mm2) and peripheral corneas (447.9±74.5) on day 1 following local application of pDCs. Flow cytometry showed a 1.4-fold increase in the density of NGF+ cells on day 3 following adoptive transfer of pDCs, compared with PBS control. One-time adoptive transfer of pDCs was accompanied by enhanced nerve regeneration on day 14 post-trephination in both the center (44.5±10.1 mm/mm2) and periphery (75.9±10.9) of corneas, compared with transfer of CD11b+ cells (24.9±11.7, p=0.02 in center and 47.7±8.2, p=0.002 in periphery) as well as PBS controls (22.2±6.3, p=0.005 in center and 62.3±4.0, p=0.04 in periphery). In corneas treated with local pDC transfer, we observed no significant increase in the density of MHC-II expressing leukocytes in the center (188.3±32.1 cells/mm2 vs. 246.4±61.4 in PBS-treated and 301.7±68.2 in CD11b+ celltreated) or periphery (205.4±24.4 vs. 250.8±18.3 in PBS-treated and 239.8±23.8 in CD11b+ celltreated) compared with control groups (p>0.05), suggesting safety of local pDC adoptive transfer.

CONCLUSIONS: Local adoptive transfer of pDCs can enhance corneal nerve regeneration following nerve damage and may serve as a novel cell-based therapeutic approach to treat corneal nerve damage.

FUNDING: NIH-R01- EY022695 (PH), NIH-R21- EY025393 (PH).

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Harvard Department of Ophthalmology

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In vitro Reconstructed Human Corneal Tissue Model: Applications to Dry Eye, Corneal Wound Healing, and Ophthalmic Drug Delivery Yulia Kaluzhny1,*, Miriam Kinuthia1, Thoa Truong1, Allison Lapointe1, Patrick Hayden1, Mitchell Klausner1

MatTek Corporation, Ashland, MA

1

*Email: ykaluzhny@mattek.com PURPOSE: The corneal barrier is vitally important for protection from environmental stress, but it also presents a major challenge for delivery of ophthalmic drugs. Current methods to investigate pathogenesis of dry eye disease (DED), corneal wound healing, biocompatibility, and permeation of topically applied ophthalmics utilize cell cultures or animals. We evaluated an applicability of the physiologically relevant, human 3D organotypic corneal tissue model for ocular research and drug development.

METHODS: Reconstructed tissues were characterized by histology, confocal microscopy, barrier function, and expression of genes essential for metabolism, detoxification, and drug transport.

Oxidative stress was generated by exposing the tissues to non-toxic doses of UV irradiation, hydrogen peroxide or by placing tissues under desiccating stress conditions (DSC) that stimulate morphological, cellular, and molecular changes relevant to DED. Corneal wounds were introduced by mechanical abrasion or alkali application and wound healing was analyzed by TEER, histology, and confocal microscopy. Corneal permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients.

RESULTS: The tissues express tight junctions, mucins, key corneal detoxification and P450 enzymes, efflax and uptake transporters. UV exposure resulted in 60X increase in ROS accumulation. 24h incubation at DSC induced phospholipid oxidation, IL-8 release, and gene up-regulation for pro-inflammatory cytokines and enzymes. The topical application of lubricant eye drops improved tissue morphology and barrier function. Upregulation of Cyclin D1 was observed after 24 hr post-abrasion and EGFR inhibitor inhibited wound healing. Application of formulations containing â&#x2030;Ľ0.02% BAC significantly reduced tissue barrier and viability after 30 min. The correlation of permeation coefficients to excised animal corneas for model drugs was r2=0.84.

CONCLUSIONS: The results demonstrate that the in vitro organotypic human corneal tissue model, EpiCorneal, structurally and functionally reproduces corneal wound healing, oxidative stress, DED markers, and is suitable for biocompartability testing and eye permeation studies. The model will avoid species extrapolation and will provide comprehensive means to study and develop ocular pharmaceuticals.

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30th Biennial Cornea Conference

Effect of Guttae-Induced Microenvironment on Corneal Endothelial Cell Behavior and Survival in Fuchs’ Endothelial Corneal Dystrophy Viridiana Kocaba1, Kishore Reddy Katikireddy1, Ilene Gipson1, Marianne O. Price2, Francis W. Price2, Ula V Jurkunas1

26

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Price Vision Group, Indianapolis, IN

1 2

PURPOSE: The number and size of guttae increase with time in Fuchs’ endothelial corneal dystrophy (FECD). This study aimed to determine the effect of guttae on corneal endothelial cell function.

METHODS: In an in vitro model, cells from a human corneal endothelial cell line, HCEnC-21T, were seeded on decellularized normal (n = 30) and FECD (n = 70) endothelial basement (Descemet) membranes (DMs). Cell shape, growth, migration, and expression of endothelial-to-mesenchymal transition (EnMT) proteins in the cells grown on the normal and FECD DMs with guttae of different diameters were assessed.

RESULTS: Cells covered a significantly greater area (97.7 ± 8.5%) on the normal DMs compared to the FECD DMs (72.8 ± 11%, p = 0.02), with a significantly greater mean number of cells (2,083 ± 153/mm2) on the normal DM than on the FECD DM (1,541 ± 221/mm2, p = 0.01). Different growth of the endothelial cells over guttae was observed on FECD DMs, depending on the guttae diameter. Guttae with a 10.51 ± 2.95 µm diameter did not impede cell growth, whereas those with a 21.11 ± 4.87 µm diameter were covered only by the cell cytoplasm with the nuclei remaining on the periphery. Guttae with the largest diameter, 31.80 ± 3.82 µm, were not covered by cells, which instead surrounded them in a rosette pattern. Cells surrounding the large guttae showed a significant upregulation of EnMT markers (actin, N-cadherin, and Snail 1) and exhibited a greater number of terminal deoxynucleotidyl transferase (TdT) deoxyuridine 5’-triphosphate (dUTP) nickend labeling (TUNEL)-positive cells, indicative of increased apoptosis, compared to any other group.

CONCLUSIONS: These findings highlight the important role of guttae in endothelial cell growth, migration, and survival. These data suggest that cell therapy procedures in FECD should be guided by the diameter of the host guttae.

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Harvard Department of Ophthalmology

27

Oral Valganciclovir Treatment for Herpes Simplex Keratitis in Patients Unresponsive to Conventional Antiviral Therapy Neslihan Dilruba Koseoglu1, Benjamin R. Strauss1, Pedram Hamrah1,*

Tufts Medical Center, Department of Ophthalmology, Boston, MA

1

*Email: phamrah@tuftsmedicalcenter.org PURPOSE: To describe four immunocompetent patients with recurrent herpes simplex virus (HSV) keratitis unresponsive to conventional antiviral therapy that improved with oral valganciclovir treatment BACKGROUND: Herpes simplex keratitis is one of the leading causes of blindness in developed countries. Treatment in a timely manner and adequate prophylaxis is crucial for preservation of vision. The current treatment options include a variety of nucleoside analogues; such as oral acyclovir, valacyclovir, penciclovir, famciclovir and topical ganciclovir, as well as DNA synthesis inhibitors such as foscarnet and cidofovir. Failure with conventional therapies in HSV keratitis can be challenging and result in permanent vision loss.

METHODS: Retrospective case series of four patients with HSV keratitis treated with oral valganciclovir between March 2016 and August 2017.

RESULTS: We reviewed the records of four patients with recurrent HSV keratitis. Three patients had a history of HSV keratitis and were on antiviral prophylaxis. One patient had a history of Bellâ&#x20AC;&#x2122;s palsy and developed HSV keratitis after cataract surgery. Three patients that presented with dendritiform epithelial keratitis were initially treated with acyclovir, valacyclovir or famciclovir for 4 to 6 months with persistent recurrences. Recurrences and failure to treatment led us to the conclusion of a possible drug resistance. We initiated oral valganciclovir 900 mg BID for 10 days as a treatment dose followed by 900 mg daily for prophylaxis. The fourth patient presented with recurrent dendritiform epithelial keratitis after discontinuing the prophylactic valacyclovir due to an allergic reaction. The patient was initially treated with famciclovir but switched to valganciclovir 450 mg daily in the second week due to an allergic reaction as well. The epithelium healed in the first two weeks in all four patients. The mean time of follow up for patients on prophylaxis was 8 months (range: 5-12 months) and none of the patients presented with any further recurrences.

CONCLUSIONS: In case of failed treatment with conventional antiviral therapies, oral valganciclovir can present a good alternative for the treatment and prophylaxis of herpetic keratitis.

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30th Biennial Cornea Conference

Neutrophil Maturation and Responses to Infectious Pathogens are Regulated by Microbiota Abirami Kugadas1, Jennifer Geddes-McAlister2, Mihaela Gadjeva1

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Division of Infectious Diseases, Department of Medicine, Brigham and Womenâ&#x20AC;&#x2122;s Hospital, Harvard Ophthalmology, Boston; 2Experimental Systems Immunology, Max Planck Institute of Biochemistry, Martinsried, Germany 1

PURPOSE: It has long been considered that neutrophils are cells with prepacked anti-microbial granules whose responses to various infectious challenges are innately pre-determined. Here, we provide data that clearly demonstrate the definition of neutrophil transcriptomes and proteomes modulated by the microbiota.

METHODS: Using RNAseq and quantitative proteomic approaches we defined mature neutrophil transcriptome and proteome signatures at steady state and during ocular infection with Pseudomonas aeruginosa. Following in-solution digestion and LC-MS/MS analysis, we quantified 3585 proteins from mature neutrophils derived from either nonâ&#x20AC;&#x201C;infected or infected germ free (GF) and specific pathogen free (SPF) mice.

RESULTS: We found that the proteomic signatures of mature neutrophils derived from the GF and SPF were significantly different at steady state. Similarly, RNAseq analysis revealed significant differential expression of 327 genes at steady state. During infection with P. aeruginosa, GFderived neutrophils responded with significant alterations in transcriptional regulation, ncRNA processing, rRNA metabolic processes, mRNA processing, and splicing, while the SPF-derived neutrophils responded with alterations in acetylation. Consistently, GF mice were more susceptible to ocular keratitis compared to SPF controls.

CONCLUSIONS: Our data support the concept that microbiota drives neutrophil maturation by defining not only the quantity, but also the quality of mature neutrophils. Based on our data, we predict that neutrophil responses can be diverse and specifically tailored to pathogens. In conclusion, neutrophil responses, although innately determined, are constantly adapted and molded by the commensal presence.

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Harvard Department of Ophthalmology

29

Purinoreceptor Activation Drives Collective Cell Migration During Epithelial Wound Repair Via Atp-Mediated Atp Release Yoonjoo Lee1,*, Gregory Teicher1, Sung Jun Son1, Vickery Trinkaus-Randall1

Departments of Biochemistry, Pharmacology and Experimental Therapeutics, and Ophthalmology, Boston University School of Medicine, Boston, MA and University of Massachusetts, Amherst, MA. 1

*Email: yjklee@bu.edu PURPOSE: Corneal injury, accompanied by improper wound repair, is the 4th-highest cause of preventable blindness, according to the World Health Organization. Our previous studies demonstrated that upon corneal injury, cells release nucleotides that activate purinergic receptors P2Y2 and P2X7. We observed that Ca2+ oscillations are sustained after injury and correlate with epithelial sheet motility. This led us to hypothesize that the presence of sustained calcium oscillations at the leading edge may regulate collective migration.

METHODS: For our calcium imaging experiments, we used human corneal-limbal epithelial cells (HCLE), a corneal cell line. HCLE were cultured to confluence on glass dishes. In order to visualize the change in calcium, cells were pre-loaded with 5 μM Fluo-3AM, a fluorescent dye, at a final concentration of 1% (v/v) DMSO and 0.02% (w/v) pluronic acid for 20 minutes at 37°C and 5% CO2. Images were collected every 3 seconds on a Zeiss Axiovert LSM 880 confocal microscope.

For image analysis we used FIJI/ImageJ and MATLAB programs.

RESULTS: After wounding, Ca2+ travels along the wound margin and to a lesser degree at regions away from the wound. The Ca2+ mobilization in cells correlate with the epithelial cells’ lamellipodial ruffling, which are more active in cells at the leading edge. Apyrase inhibits the secondary response indicating a need for extracellular ATP, and there is a unique profile of activation for the P2Y2 and P2X7 receptors. Cluster and % intensity analysis performed after stimulating each of the purinergic receptors on confluent cells demonstrated that P2Y2 has a higher synchronicity and correlation between cells than those of P2X7.

CONCLUSIONS: Together, these results led us to speculate that the two receptors mediate specific phases of collective migration and generate extensive cell-cell communication along wound margin. Our work describes a novel important function for the secondary waves in generating collective migration.

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30th Biennial Cornea Conference

Inverse Spectroscopic Optical Coherence Tomography and Two-Photon Autofluorescence Imaging Applied to the Evaluation of Ocular Surface Lesions

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Hyunjoo J. Lee1, Lei Zhang2, Sharmila Masli1, Ji Yi2 Department of Ophthalmology, Boston University School of Medicine, Boston, MA; Medicine, Boston University School of Medicine, Boston, MA

1 2

PURPOSE: The ability to diagnose ocular surface neoplasia with the aid of imaging modalities is improving, but still hindered by the limitations of currently available techniques. Inverse spectroscopic optical coherence tomography (IS-OCT) is an emerging technique capable of detecting nanoscale ultrastructural differences in pre-neoplastic and neoplastic states within gut mucosa. Two-photon autofluorescence imaging (TPI) can detect autofluorescence of metabolic markers: nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), alterations in which have been associated with neoplasia. In this pilot study, we investigated the utility of IS-OCT and TPI in diagnosing lesions of the ocular surface by evaluating excised conjunctival lesions.

METHODS: With IRB approval, informed consent was obtained from patients undergoing ocular surface lesion excision at Boston Medical Center by a single surgeon. The surgeon used a 2 mm biopsy punch to separate a central portion of the excised lesion, leaving the lateral margins intact, and sent the peripheral portion directly to the pathology lab and the central portion for ex vivo IS-OCT and TPI. Immediately after imaging, the central specimen was fixed and also sent to the pathology lab for histological diagnosis. The IS-OCT and TPI findings were correlated to the histologic findings.

RESULTS: 11 specimens were collected. Based on histologic findings, 1 specimen was a conjunctival papilloma with dysplastic features, 1 was a squamous cell carcinoma (SCC) in situ, 1 was limbal tissue with stromal fibrosis (from a map biopsy taken from a previous site of melanoma in situ), and 8 were pterygium. An IS-OCT quantitative measure correlating to ultrastructural compaction, D value, was higher in SCC (4.38, 2.6-fold), papilloma (3.73, 2.2-fold) and tissue from a site of previous conjunctival melanoma (2.92, 1.75-fold), compared to pterygium (1.664 Âą0.30).

NADH autofluorescence by TPI was highest in the papilloma (25.98, 5.5-fold) and SCC (14.22, 3-fold) compared to pterygium (4.77Âą 1.33).

CONCLUSIONS: Compared to pterygium, lesions with higher malignant potential had higher D and higher NADH autofluorescence. We demonstrate proof of concept that IS-OCT and TPI can be applied to the examination of ocular surface lesions.

91

Harvard Department of Ophthalmology

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Mast Cells Contribute to the Induction of Corneal Alloimmunity Mingshun Li1,*, Anuradha Sahu1, Sharad Mittal1, Srikant Sahu1, Afsaneh Amouzegar1, Sunil Chauhan1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

Email: Mingshun_Li@meei.harvard.edu

*

PURPOSE: To investigate whether ocular surface mast cells contribute to allosensitization and graft rejection using a mouse model of corneal transplantation.

METHODS: Allogeneic transplantation was performed by grafting donor cornea (C57BL/6 mice) onto recipient bed in BALB/c mice. To examine the effect of transplantation on mast cells, the frequencies of c-Kit+ FCεR1+ mast cells and their expression of TNFα were measured in cornea and conjunctiva of transplant recipients using flow cytometry. β-hexosaminidase assay was performed to evaluate mast cell activation. To determine the effect of mast cell activation on allosensitization, graft recipients were topically treated with cromoglycate (a mast cell inhibitor) or PBS 3 times a day for 2 days. Corneas and draining lymph nodes (DLN) were harvested to evaluate infiltration and activation of MHCII+CD11b+ mature antigen presenting cells (APCs), CD11b+ macrophages and CD4+IFNγ+ Th1 cells using flow cytometry. Allograft survival was evaluated weekly by slit-lamp microscopy for 8 weeks.

RESULTS: There was a significant 2-fold increase in the frequencies of mast cells (p<0.01) and ~ 50% increase in the expression levels of β-hexosaminidase and TNFα by mast cells in transplanted mice compared to normal controls (p<0.05). Topical application of mast cell inhibitor in transplant recipients resulted in significantly 40% reduced inflammatory cell infiltration (p<0.01) and 35% decreased maturation of APCs in the cornea compared to PBS-treated controls (p<0.05).

Moreover, inhibition of mast cells led to a significant 80% reduction in the generation of graft attacking Th1 cells in DLNs (p<0.001) and their infiltration into the graft, leading to a substantially prolonged corneal allograft survival compared to PBS-treated recipients.

CONCLUSIONS: Our data demonstrate that significant mast cell activation occurs during transplantation. Moreover, early inhibition of mast cell function leads to decreased allosensitizaton and prolonged allograft survival.

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30th Biennial Cornea Conference

Regional Analysis of Lid Wiper Epitheliopathy Detected by Vital Staining of the Eyelid Margin in Patients with Dry Eye Disease Secondary to Meibomian Gland Dysfunction

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Michael E. Lindsay1, Deidra M. Soto1, Paula J. Oliver1, Jack V. Greiner1 Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

PURPOSE: The purpose of this study was 1) to characterize lid wiper epitheliopathy (LWE) of the eyelid margin in patients with meibomian gland dysfunction and 2) to compare regional analyses of LWE in the upper and lower eyelids.

METHODS: Patient selection criteria included validated Standard Patient Evaluation for Eye Dryness questionnaire scores of â&#x2030;Ľ6 and meibomian gland assessment (MGA) scores of â&#x2030;¤12 measured on the lower eyelid (n=43 eyes). The upper and lower eyelid margins were each divided into four regions. The far nasal region (FNR) extended from the nasal canthus to the punctum. The remaining regions comprised that portion of the eyelid margin over the tarsal plate divided into 3 equidistant zones (temporal, central, nasal). The degree of epitheliopathy as detected by Lissamine green (LG) staining was graded by area of staining on a scale from 0 to 3, with 0 indicating only normal staining of the Line of Marx (ML) up to 3 indicating severe staining extending more than 1 mm posterior to the ML. RESULTS: The LWE in the FNR of the lower eyelid stained to a greater degree than the FNR of the upper eyelid (p<0.0006). Comparing the FNR to the average LG grade of the remaining regions of the eyelid, there were differences in both upper (p<0.0001) and lower eyelids (p<0.0001). In contrast, there were no inter-region differences in LG staining among the temporal, central or nasal regions of the upper lid (p>0.05 for all comparisons) or the lower lid (p>0.05 for all comparisons), nor was there any difference between the composite (temporal, central, nasal) scores comparing the upper and lower eyelids (p=0.48).

CONCLUSIONS: This study presents three unexpected findings: 1) LWE staining is present in the FNR, and to a greater degree than the rest of the eye lid margin over the tarsal plate. 2) The lower FNR has greater LWE staining than the upper FNR. 3) And the degree of LWE staining is the same in upper and lower eyelids even though blink movement is much greater in the upper lid and, thus, might have greater staining. Considering these observations in the context of eyelid blink dynamics, it appears that LWE may not only result from lid wiping trauma, but also from dehydration of the mucous membrane, perhaps related to incomplete closure of the eyelid, especially in the FNR.

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Harvard Department of Ophthalmology

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Biomarkers for Progenitor and Differentiated Epithelial Cells in the Human Meibomian Gland Yang Liu1, Hua-Tao Xie1,2, Mark P. Hatton1,3, Wendy R. Kam,1 David A. Sullivan1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Union Hospital, Huazhong University of Science and Technology, Wuhan, China; 3Ophthalmic Consultants of Boston, Boston, MA 1 2

PURPOSE: The meibomian gland (MG) is a sebaceous gland that secretes through a holocrine process. Because such secretion requires the destruction of MG acinar epithelial cells, they need constant renewal and differentiation. The processes that promote these regenerative events in the human MG are unknown. Indeed, it is not even known how to distinguish MG progenitor and differentiated cells. Such identifying ability would have tremendous value in clinical efforts to restore MG function, and to regenerate MGs after disease-induced dropout. We hypothesize that Lrig1 and DNase2 serve as biomarkers for human MG progenitor and differentiated cells, respectively.

Our rationale is that Lrig1, a master regulator of epithelial stem cells, is a biomarker for progenitor cells that give rise to differentiated sebaceous gland epithelial cells (sebocytes). Further, lysosomal DNase2 is known to initiate sebocyte holocrine secretion, the terminal step of differentiation. Our study was designed to test this biomarker hypothesis.

METHODS: We utilized human lid tissues and immortalized human meibomian gland epithelial cells (IHMGECs). The use of human tissues was approved by the Mass. Eye and Ear IRB. Samples were processed for immunofluorescent and Western blotting procedures.

RESULTS: Lrig1 is expressed in MG basal epithelial cells in the acinar cell periphery, a location in which progenitor epithelial cells originate in sebaceous glands. Lrig1 is not expressed in the differentiating cells in the central section of the MG acinus. In contrast, DNase 2 is expressed in the differentiated epithelial cells of the MG central acinus, but not in basal cells. We also discovered that proliferation stimulates, and differentiation suppresses, Lrig1 expression in IHMGECs. The opposite is true for DNase 2 expression.

CONCLUSIONS: These results support our hypothesis and demonstrate that Lrig1 and DNase2 serve as biomarkers for progenitor and differentiated cells in the human MG. FUNDING: This research was supported by the Margaret S. Sinon Scholar in Ocular Surface Research Fund, and the Guoxing Yao Research Fund.

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30th Biennial Cornea Conference

UVA-induced Mouse Model of Fuchs’ Endothelial Corneal Dystrophy is Mediated by DNA Damage and Is Exacerbated in Females Cailing Liu1, Takashi Miyai1,Taiga Miyajima1; Shivakumar Vasanth1, Dijana Vojnovic1, Bill A. Farinelli2, Ula V.Jurkunas1

34

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA

1 2

PURPOSE: Fuchs’ endothelial corneal dystrophy (FECD) is characterized by progressive loss of corneal endothelial cells (CEnC) with a higher prevalence in women. In this study, we aimed to establish an in vivo model of late-onset and age-related FECD by inducing oxidative stress via UVA irradiation of mouse CEnCs (MCEnCs) and further investigate the role of oxidative stress in FECD pathogenesis.

METHODS: Eyes of 7-15-week-old male and female C57BL/6 mice were irradiated with UVA with a single fluence of 250 J/cm2, 500 J/cm2, 750 J/cm2 or 1000 J/cm2. MCEnC morphology and density were examined by HRT and corneal thickness (CCT) was measured by OCT at 1 d, 2 d, 3 d, 1 wk, 2 wk, 1 mo, 2 mo, and 3 mo post UVA irradiation. Contralateral eyes without UVA irradiation served as controls. Corneal whole mounts from UVA-irradiated and control eyes were analyzed with anti-ZO-1 antibody, 8-OHdG antibody and TUNEL staining. Corneal endothelium with Descemet’s membrane (DM) was examined by transmission electron microscopy (TEM).

Relative expression of LC3A/B was analyzed by Western blotting.

RESULTS: UVA irradiation resulted in progressive morphological alterations of MCEnC, reduction in cell density, and increased polymegathism and pleomorphism in a time- and dose- dependent manner. Specifically, HRT revealed dark areas of lost MCEnCs surrounded by disrupted cell boundaries, suggestive of findings observed in FECD patients. Moreover, UV-A led to an acute increase in CCT that peaked during 2- 7 days post mice irradiated. CCT recovered between 7-60 days post UVA, but exhibited a second wave of increase at 2 months post UVA, indicating a chronic phase of endothelial dysfunction. Importantly, with same dose of UVA, females showed greater alterations in MCEnC morphology and in CCT as compared to males, indicating females more susceptible to UVA as compare with males. At 3 months post 1000 J/cm2 UVA, DM thickness increased 1.3 fold in UVA-treated eyes as compared to no-UVA treatments. TEM showed ballooned cristae and extensive vacuolization in UVA treatments as compared to controls.

UVA irradiation elevated the levels of H2O2 in aqueous humor, TUNEL and 8-OHdG staining in MCEnCs. In addition, UVA led to increase in autophagy marker LC3A/B specifically in females at 1 day post UVA. CONCLUSIONS: Exposure of mouse corneas to UVA resulted in a progressive loss of MCEnC regular hexagonal shape and cell density. Loss of endothelial morphology was accompanied by an increase in CCT, DM thickness, and cell apoptosis. The morphological and functional changes induced by UVA were exacerbated in females recapitulating female predominance observed in FECD. This study provides the first animal model and evidence that oxidative stress plays an important role in late-onset and age-related FECD pathogenesis.

95

Harvard Department of Ophthalmology

35

AAV Gene Therapy in a Canine Model of MPS1 Prevents and Reverses Corneal Blindness Telmo Llanga2, Keiko Miyadera3, Laura Conatser2, Brian C. Gilger 4, Richard Davis1, Joanne Kurtzberg5, Richard J. Samulski 2, Matthew Hirsch1,2

Ophthalmology, University of North Carolina, Chapel Hill, NC; 2Gene Therapy Center, University of North Carolina, Chapel Hill , NC; 3University of Pennsylvania, Philadelphia, PA; 4North Carolina State University, Raleigh, NC; 5Duke University, Durham, NC 1

PURPOSE: Mucopolysaccharidosis type 1 (MPS1) is a progressive lysosomal storage disease that can be lethal in the first decade of life. Cell Therapy through bone marrow transplantation is able to prolong life by decades, however, non-lethal MPS1 manifestations significantly decrease the patientâ&#x20AC;&#x2122;s quality of life. In particular, approximately 90% of MPS1 patients present corneal opacity with about 50% of cases leading to complete vision loss. There is currently no effective treatment for MPS1 corneal blindness, which is the deficit we seek to address in this work using a viral based gene addition strategy.

METHODS: A codon optimized IDUA cassette was packaged in a chimeric AAV capsid identified efficient for cornea transduction. Previous evaluations in human corneas ex vivo demonstrated supraphysiological IDUA activity and safety following intrastromal injection of AAV8G-optIDUA. These optimistic data warranted an efficacy study in naturally occurring MPS1 canines that also suffer from corneal clouding. Three canines with advanced (13 m.o.) and one with early (9 m.o.) corneal clouding were administered AAV8G9-GFP (negative control) in one eye and AAV8G9optIDUA in the contralateral eye via cornea stromal injections. These canines were monitored by slit lamp biomicroscopy and corneal imaging, blood samples taken before and after treatment, as well as postmortem tissue examination, evaluating total area of polysaccharide accumulation by Alcian Blue histological Staining, central corneal thickness, morphology and infiltration by H&E histopathology veterinary-physician scoring, automated corneal vascularization quantification via ImageJ, therapy induced neutralizing antibody response, and vector bio-distribution evaluated by qPCR. RESULTS: Corneal clearing, encompassing both the actual cloudy storage disease and regression of associated vascularization was apparent as early as 1 week and near complete disease reversal was evident as early as 13 weeks in all corneas injected with AAV-8G9-optIDUA in the symptomatic cohort. In the younger animal with the early disease, corneal disease progression continued in the control eye while the AAV8G9-optIDUA injected cornea demonstrated clearance of microscopic storage materials and did not develop vascularization. No adverse effect was observed specific to the injection or either of the transgenes, except for transient reversible corneal edemas that developed in each eye of the animal injected at the highest viral dose. This same animal was also the only one to develop neutralizing antibodies to the viral vector capsid. Vector Biodistribution of IDUA was present mainly in eye structures, with minimal dissemination outside the eye.

CONCLUSIONS: The collective data of a corneal gene addition strategy using AAV vectors for MPS1 blindness demonstrate safety and efficacy and lend support for the translation of this technology to human MPS1 patients.

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Detection of O-Glcnac-Modified Proteins in Human Ocular Surface Epithelial Cells Nicole M. McColgan1,*, Pablo Argüeso1

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Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

*Email: nicole_mccolgan@meei.harvard.edu PURPOSE: O-linked β-N-acetylglucosamine, termed O-GlcNAc, is a common carbohydrate modification of serine or threonine residues found on nuclear, mitochondrial, and cytoplasmic proteins. It plays important roles as a nutritional sensor and in mediating physiological and pathological processes. This study investigated the presence of O-GlcNAc in human ocular surface epithelial cells.

METHODS: Immortalized human corneal-limbal epithelial cell cultures were grown as monolayers and in a differentiated stratified cell culture system. The presence of O-GlcNAc-modified proteins was determined in cell lysates and digitonin extracts using the CTD110.6 monoclonal antibody.

Cells were supplemented with GlcNAc or PUGNAc, an inhibitor of the O-GlcNAc hydrolase.

Expression levels of O-GlcNAc transferase in conjunctival impression cytology samples from normal and dry eye patients were analyzed using a glycogene microarray database.

RESULTS: A similar pattern of O-GlcNAc-modified proteins was found in monolayer and differentiated corneal epithelial cell cultures at low–, medium– and high–molecular weights. The cytosolic fractions derived from digitonin permeabilization exhibited a unique pattern of bands with different intensities compared to the remaining subcellular components. Supplementation with GlcNAc and inhibition of O-GlcNAc hydrolase resulted in the activation of the hesoxamine pathway as shown by increased O-GlcNAc levels in the cytosol. Microarray analysis of impression cytology samples demonstrated a significant decrease in O-GlcNAc transferase mRNA levels in dry eye patients compared to normal subjects (p<0.001).

CONCLUSIONS: Data from this study suggest that the hexosamine biosynthetic pathway can be activated by GlcNAc supplementation in human corneal epithelial cells which could have therapeutic implications in ocular surface disease.

FUNDING: NIH Grant R01EY026147

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Harvard Department of Ophthalmology

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Predicting Ocular Irritancy using an In Vitro Corneal Tissue Model Tina B. McKay1,*, Siran Wang1, Chiara Ghezzi1, Sophia Bunnell1, David Kaplan1

Department of Biomedical Engineering, Tufts University, Medford, MA

1

*Email: tina.mckay@tufts.edu PURPOSE: Regulatory agencies require screening of commercial products for ocular toxicity prior to marketing in the United States. Due to the high-volume of chemicals continually in development, a shift from animal testing to more high-throughput, reliable cell culture methods have arisen over the years in an effort to “reduce, refine, and replace” the use of animals in assessing chemical safety and efficacy. Our current study assessed the effectiveness of our corneal tissue model to evaluate and predict ocular irritancy based on epithelial, stromal, and neuronal interactions that occur in vitro.

METHODS: Using a co-culture system of primary human cells of corneal epithelium, keratocytes, and sensory neurons, we have bioengineered an in vitro model of the innervated cornea using functionalized silk scaffolds. Irritant application was performed on mature constructs (4 weeks) with short exposure (10min) followed by observation at t=24hrs and 7 days. We defined the following endpoints as primary measures of ocular irritancy: 1) tissue permeability (fluorescein staining), 2) tissue opacity (visible light transmittance), 3) cell viability (MTT assay), 4) neuropeptide release (substance P and CGRP), and 5) inflammatory cytokine secretion (IL-1, TNF-α). We compared our results to an ex vivo assay utilizing isolated porcine corneas.

RESULTS: In agreement with Draize scores, our corneal model was susceptible to loss in corneal transparency and increased permeability following severe irritant application. Substance P secretion was increased with moderate irritants but reduced with severe irritants consistent with cell toxicity.

Secretion of pro-inflammatory factors were elevated by select chemicals (capsaicin, trichloroacetic acid, and triton-X-100), but reduced with organic stimulants (n-octanol and menthol) supporting the role of TRPV1 activation in driving hyperalgesic pathways with exclusion of chemicals that may permeate the cell membrane directly. In contrast, the ex vivo assay was most effective at predicting ocular toxicity for chemicals with potent corrosive properties but failed to accurately assess chemicals that may lead to less severe irritation.

CONCLUSIONS: Our corneal tissue model integrating multiple cell types (epi, stroma, neuronal) is effective at discerning more subtle effects of chemical application on inducing ocular irritancy and may serve as an appropriate in vitro method to study ocular pain mechanisms.

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30th Biennial Cornea Conference

Stromal Cells Regulate Ocular Inflammation by Suppressing Neutrophil Effector Functions Sharad Mittal1,*, Anuradha Sahu1, Afsaneh Amouzegar1, Mingshun Li1, Sunil K. Chauhan1

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Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

Email: Sharad_Mittal@meei.harvard.edu

*

PURPOSE: Neutrophil-secreted effector molecules, myeloperoxidase (MPO) and N-elastase (ELAN), are the primary cause of tissue damage during corneal inflammation. In the present study, we have investigated the effect of stromal cells in regulating the secretion of neutrophil effector molecules and subsequent tissue damage after corneal injury.

METHODS: To investigate the effect of stromal cells on neutrophil effector functions in vitro, bone marrow purified non-hematopoietic stromal cells and fMLP-activated neutrophils were co-cultured in the presence or absence of transwell inserts for 1 hour. Neutrophil effector molecules, MPO and ELAN, were quantified at protein levels using ELISA. To determine the in vivo effect of stromal cells on neutrophil function, corneal injury was created by mechanical removal of the corneal epithelium and anterior stroma. Stromal cells were intravenously injected into mice 1 hour after induction of injury. Corneas were harvested to evaluate MPO expression and ocular infiltration of CD11b+Ly6G+ neutrophils using flow cytometry. Immunohistochemistry was performed to evaluate corneal tissue structure.

RESULTS: Activated neutrophils co-cultured with stromal cells showed a significant 2-fold decrease in secretion of MPO and ELAN compared to neutrophils activated alone (p<0.05). This suppressive effect was cell-cell contact dependent, as stromal cells co-cultured with neutrophils in the presence of transwell failed to suppress the secretion of neutrophil effector molecules.

Following corneal injury, stromal cell-treated mice showed significant 40% decrease in the expression of MPO by neutrophils and lower neutrophil frequencies at the ocular surface compared to the untreated injured controls (p<0.05). Reduced expression of MPO by neutrophils was also accompanied by normalization of corneal tissue structure following stromal cell treatment.

CONCLUSIONS: Stromal cells inhibit neutrophil effector functions via direct cell-cell contact interaction during inflammation. Results from this study could have implications in the treatment of inflammatory ocular disorders caused by excessive neutrophil activation.

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Harvard Department of Ophthalmology

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Corneal Plasmacytoid Dendritic Cells Play a Tolerogenic Role in Corneal Transplantation Hamidreza Moein1,3, Maryam Tahvildari3, Arsia Jamali1,3, Maria J. Lopez1,3, Deshea L. Harris1,3, Reza Dana3, Pedram Hamrah1,2,3

Center for Translational Ocular Immunology; 2Cornea Service, New England Eye Center and Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA; 3Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA 1

PURPOSE: Corneal allograft rejection is induced by adaptive immune responses. The purpose of this study was to investigate the role of corneal plasmacytoid dendritic cells (pDCs) in mitigating allosensitization and inducing tolerance to corneal allografts.

METHODS: Wild type (WT) BALB/c (H-2d) mice were used as donors. Transgenic BDCA-2DTR mice on C57BL/6 (B6; H-2b) background, in which local pDCs can be selectively depleted with subconjunctival (s.cj.) diphtheria toxin (DT) injections were used as recipients. WT B6 mice were used as sham controls. DT (30 ng) was injected s.cj. one day before transplantation and was continued every other day for two weeks. Graft opacity was evaluated up to 2 weeks after transplantation using a standard scale. Graft infiltration of CD45+cells (pan-leukocyte marker), CD68+macrophages and CD4+T cells were measured using flow cytometry. Ipsilateral draining lymph nodes (dLN) were assessed for host allosensitization using enzyme-linked immunospot (ELISPOT) assay and frequencies of IFNγ+CD4+T cells, IL17+CD4+T cells and CD25 expression levels in CD4+CD25+Tregs using flow cytometry.

RESULTS: Mean opacity scores were significantly higher in pDC-depleted mice compared to the sham controls at day 7 (3.7/5 vs. 1.33/5; p<0.001) and day 14 (4.6/5 vs 3.41/5; p=0.027) post-transplantation. Infiltration of CD45+cells in corneal grafts was significantly higher in the pDC-depleted group vs. controls at day 7 (46.3% vs. 28.6% of total graft cells; p<0.01), including CD68+macrophages (59.07% vs. 33.4%; p=0.0005) and CD3+CD4+T cells (9.93% vs. 2.5%, p<0.0001). ELISPOT analysis showed increased IFNγ+T cells in both direct (p=0.026) and indirect (p=0.016) pathways of allosensitization in the pDC-depleted group vs. controls. Flow cytometric analysis of dLNs showed increased frequencies of CD4+IFNγ+T cells (2.2% vs. 0.95%; p=0.007) and CD4+IL17+T cells (2.87% vs. 1.67%; p=0.046) in the pDC-depleted hosts vs. sham controls.

There was a significant decrease in mean expression of CD25 among Tregs in the pDC-depleted group vs. sham controls (mean fluorescent intensity: 211 vs. 154, p=0.04).

CONCLUSIONS: Depletion of pDCs in the cornea results in increased host allosensitization and accelerated leukocyte graft infiltration. These results demonstrate the capacity of pDCs to mediate T cell responses, and provide valuable evidence for cell-mediated therapy to promote corneal transplant survival.

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Conjunctival Biopsy for Diagnosis of Ocular Cicatricial Pemphigoid Laura Eggenschwiler1, Mariantonia Ferrara1,2, Stephen D. Anesi1, Charles S. Foster1,3

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Massachusetts Eye Research and Surgery Institution, Waltham, MA; 2Department of Neuroscience, Reproductive and Odontostomatological Science, Federico II University, Naples, Italy; 3Harvard Ophthalmology, Boston, MA 1

PURPOSE: To demonstrate the reliability of conjunctival biopsy analyzed by direct immunofluorescence (DIF) and supplemented with avidin-biotin complex immunoperoxidase (ABC), as needed, in the diagnosis of ocular cicatricial pemphigoid (OCP).

METHODS: This is a retrospective study. We reviewed the results of 136 consecutive patients conjunctival biopsies at Massachusetts Eye Research and Surgery Institution (MERSI) from 2008 to 2016 due to the clinical suspicion of OCP and to report the response to therapy in patients with positive biopsy.

RESULTS: Among 136 patients, 90 (66%) were diagnosed with OCP on the basis of positive DIF and additional 18 patients by ABC immunoperoxidase. This represents an increase in sensitivity of conjunctival biopsy from 79.6% with DIF to 95.6% with supplemental ABC. Among 57 biopsypositive patients who have been followed for at least 2 years at MERSI, 44 were in remission at 1-year follow-up visit and 50 after 2 years.

CONCLUSIONS: Conjunctival biopsy analyzed by histopathology and DIF supplemented by ABC immunoperoxidase has a high reliability for the diagnosis of OCP and should be mandatory in patients with clinical suspicion of OCP before starting long-term immunomodulatory therapy. The accurate and early detection of OCP allows earlier initiation of therapy and better clinical outcomes.

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Harvard Department of Ophthalmology

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Interferon Gamma-Positive Natural Killer Cells Contribute to Corneal Allograft Rejection in Young Mice Takeshi Nakao1,*, Takenori Inomata1, Maryam Tahvildari1, Reza Dana1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

*Email: Takeshi_Nakao@meei.harvard.edu PURPOSE: Survival rates of corneal allografts in children are much lower than in adults. However, the immunologic mechanisms responsible for the higher graft failure rates in young recipients are not clear. The purpose of this study was to investigate the contribution of natural killer (NK) cells to corneal allograft rejection in young vs. adult graft recipients.

METHODS: Allogeneic corneal transplantation was performed using 3.5-week-old and 10-week-old BALB/c mice as recipients and C57BL/6 mice as donors (n=10 per group).

Weekly graft examinations were performed until 8 weeks post-transplantation to evaluate graft survival. Infiltration of NK cells (CD45+CD3-CD49b+) into the grafts was assessed at day 21 after transplantation using flow cytometry. Draining lymph nodes (dLNs) were examined to evaluate the frequencies of interferon gamma (IFNg)-positive NK cells and the expression of CD25 surface marker by NK cells in young and adult graft recipients at day 7 after transplantation.

RESULTS: 3.5-week-old mice demonstrated significantly lower survival rates compared to 10-week-old mice at week 8 after transplantation (10% vs. 50%, with median survival of 35 vs.

49 days; P=0.027). The frequencies of NK cells in the cornea were significantly higher in 3.5 compared to 10-week-old mice at day 21 post-transplantation (5.04% vs. 0.280%; P=0.0054).

In addition, the frequencies of IFNg-positive NK cells and the mean fluorescence intensity (MFI) of CD25 expression by NK cells were significantly higher in the dLNs of 3.5 compared to 10-week-old mice at day 7 post-transplantation (17.7% vs. 12.4%; P=0.0042 and MFI=1218 vs. 666; P=0.014, respectively).

CONCLUSIONS: Early rejection of corneal allografts in young graft recipients correlates with IFNgpositive NK cells.

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30th Biennial Cornea Conference

Anterior Segment Optical Coherence Tomography Angiography (AS-OCTA) as a Tool to Analyze Conjunctival Vessels and Measuring Its Density in Ocular Surface Diseases

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Ricardo M. Nosé1,2,*, Nicholas J. Pondelis1, Arsia Jamali1, Paula Kataguiri1,2, Anam Akhlaq1,2, Gabriela Dieckmann1,2, Kenneth R. Kenyon2, Pedram Hamrah1,2 Center for Translational Ocular Immunology; 2 Cornea Service, New England Eye Center, Tufts Medical Center, Tufts School of Medicine, Boston.

1

*Email: ricnose@gmail.com PURPOSE: To assess the vascular density by anterior segment optical coherence tomography angiography (AS-OCTA) in patients with ocular surface diseases, as compared to healthy eyes.

METHODS: For this retrospective study, we used the corneal adaptor module from the AngioVue system (Optovue, Fremont, California, USA) to create scans from temporal and nasal conjunctiva, using the 6x6 mm angiography imaging software. The 250 micrometers (μm) thickness images were then analyzed by the vessel density software from the same device. We included healthy volunteers (n=6 eyes), as well as patients with mild to moderate dry eye disease (n=8 eyes), contact lens wearer (n=2 eyes), and ocular allergy (n=3eyes). T-test was used to average vessel densities between nasal and temporal quadrants. General linear model followed by LSD adjustments was used to compare the groups. P < 0.05 were considered significant.

RESULTS: Results of conjunctival AS-OCTA showed that the blood vessel density in healthy eyes were 57.4% ± 1.5 temporally and 56.3% ± 1.3 nasally (p=0.20) with a mean of 56.8% ± 0.3. In contrast, we observed significant increase of the mean vessel density in patients with dry eye disease was 58.5% ± 1.6 (p=0.02). Further mean vessel density in contact lens wearer was 62.5% ± 0.8 (p=0.001) and 64.1% ± 0.9 (p<0.001) in ocular allergy compared to healthy participants.

CONCLUSIONS: OCTA is a reliable tool in detecting conjunctival vessels and measuring its density, showing an increased blood vessel density in patients with dry eye disease, contact lens wear, and ocular allergic disease. Larger studies are warranted to assess conjunctival vessel density in ocular surface disease.

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Harvard Department of Ophthalmology

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Alterations in Basal Lamina Stiffness and Vinculin Dynamics Lead to Impaired Epithelial Cell Migration Obi Onochie1,*, Celeste Rich1, Alicia Zollinger2, Michael Smith 2, Vickery Trinkaus-Randall1,3

Department of 1Biochemistry, Boston University School of Medicine; 2Department of Biomedical Engineering, Boston University; 3Department of Ophthalmology, Boston University School of Medicine 1

*Email: onochie7@bu.edu PURPOSE: Corneal injury remains the fourth cause of blindness according to the World Health Organization. Certain conditions such as diabetes and corneal infection, may cause the cornea to be exposed to a hypoxic environment. Our data indicate that corneas exposed to hypoxic environments present with alterations in the morphology of the leading edge, composition of the basal lamina, and potential changes in stiffness. We hypothesize that altering the stiffness of the basal lamina affects epithelial migration and wound healing, which may mediate signaling of focal adhesion proteins.

METHODS: Rat corneas were dissected and cultured as previously described (Lee, Albert et al, 2014). For traction force migration (TFM), corneas were dissected and incubated for 35 minutes in DMEM and 1.2 units/mL of Dispase II. Epithelial sheets were removed and placed onto Fibronectin (FN)-coated beaded gels. For immunofluorescence staining, human corneal limbal epithelial (HCLE) cells or organ cultures were incubated overnight in primary antibodies then treated with Alexa Fluor-conjugated secondary antibodies for 1 hour.

RESULTS: On FN and Collagen IV-coated polyacrylamide gels, HCLE cell morphology, size, and motility is affected by the change in substrate stiffness rather than by alterations in matrix coating.

With increased stiffness, the expression and phosphorylation of vinculinY1065 is altered in our organ culture model and in vitro system. Localization of vinculinY822 is also altered. We employed TFM to determine the distance travelled and the amount of force exerted by epithelial sheets as they migrate on substrates with varying stiffness. We observed that sheets exhibit retrograde movement prior to forward migration.

CONCLUSIONS: The results indicate that alterations in substrate stiffness may affect corneal epithelial migration. By further examining migration we aim to improve the impaired wound healing mechanisms resulting from chronic hypoxia.

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30th Biennial Cornea Conference

The Impact of Mitomycin C treatment on Corneal Epithelial Cell Adhesion, Migration, Corneal Nerve Reinnervation And Nerve Crush Injury

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Sonali Pal-Ghosh1,*, Gauri Tadvalkar1, James D. Zieske2, Xiaoqing Guo2, Mary Ann Stepp1,3 George Washington University School of Medicine and Health Sciences, Department of Anatomy and Regenerative Biology, Washington, DC; 2Harvard Ophthalmology/Mass. Eye and Ear, Boston, MA; 3George Washington University School of Medicine and Health Sciences, Department of Ophthalmology, Washington, DC 1

*Email: spghosh@gwu.email.edu PURPOSE: To determine whether Mitomycin C (MMC) treatment impacts corneal epithelial cell adhesion and intraepithelial corneal nerve (ICN) reinnervation after trephine (axon crush) injury.

METHODS: Telomerase-immortalized Human Corneal and Limbal Epithelial (HCLE) cells and Primary Human Corneal Epithelial (PHCE) cells were treated with MMC for 3 hrs or with Conditioned Media (CM) from MMC treated corneal fibroblasts and HCLE cells. Cell migration was assessed by time lapse imaging. Cell adhesion, protein synthesis, and mitochondrial numbers were also quantified. Reinnervation of the corneal sensory nerves was quantified in vehicle and MMC treated mouse corneas over time after 1.5 mm nerve crush injury.

RESULTS: Treating HCLE and PHCE with MMC for 3 hr followed by cell recovery overnight in cell culture media without MMC lead to significantly reduced cell migration as did treating cells with CM from MMC treated corneal fibroblasts or HCLE cells. Cell adhesion decreased with MMC treatment but increased in cells with CM from MMC-treated fibroblasts. Responses of HCLE and PHCE cells to direct MMC treatment differ from those in response to factors secreted by MMCtreated cells. In vivo experiments quantifying axon density after MMC treatment following nerve crush injury, show that reinnervation of the corneal nerves is delayed significantly after MMC treatment but axon density recovers 6-8 weeks after injury.

CONCLUSIONS: These data show that factors released by MMC treated corneal epithelial cells and fibroblasts increase corneal epithelial cell adhesion in vitro and transiently, in vivo, delay recovery of axon density after nerve crush wounds.

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Harvard Department of Ophthalmology

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Chemical Burns to the Cornea and Neuroglia Changes: New Therapeutic Possibilities Eleftherios I. Paschalis1,2, Fengyang Lei2, Chengxin Zhou2, Reza Dana1,2, Demetrios Vavvas2, James Chodosh2, Claes H. Dohlman2

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, MA; 2Harvard Ophthalmology/Mass. Eye and Ear, Boston, MA 1

PURPOSE: Severe bilateral chemical burns, especially from alkali, still carry a grim prognosis for final visual outcome. In addition to the corneal damage, severe glaucoma is a common complication. Lavage and corticosteroids have only moderate effect and standard keratoplasty has usually poor prognosis. This study was designed to investigate the physical and molecular mechanisms of retinal damage and to explore the role of antibody therapy for retinal neuroprotection.

METHODS: Corneal alkali burns were performed in mice and rabbits, followed by copious irrigation. Micro-electrode pH, O2 and redox sensors were implanted in the anterior and posterior segments of the eye for continuous 24-hour in vivo measurements. Craniotomies were performed in live animals and fluorogold particles were injected in the superior colliculus for retrograde labeling of viable retinal ganglion cells (RGC). Retinal inflammation was evaluated using qPCR, protein analysis and cellularly by employing transgenic and knock-out mice in conjunction with bone marrow chimeras. The roles of microglia and blood-derived monocytes in retinal injury was further assessed using pharmacological depletion with CSF1R inhibitors. TNF-α blocker (infliximab) was administered systemically (6.25mg/kg) in a single dose 15 minutes after the burn to evaluate the neuroprotective effect.

RESULTS: In our animal studies, we have shown that severe apoptosis occurs in the ganglion cell layer rapidly after an alkaline burn to the cornea. This effect is not caused by a direct pH elevation in the retina – alkali is effectively buffered at the iris-lens level – but by inflammatory cytokines generated anteriorly and rapidly diffusing posteriorly – TNF-α being one of them. Administration of TNF-α inhibitor (infliximab) was highly neuroprotective. Results were confirmed using TNF receptor knockout mice, which showed reduced inflammation and RGC protection. On the cellular level, peripheral monocytes from the blood and optic nerve were shown to enter the retina within 24 hours, releasing additional TNF-α and other pro-inflammatory mediators. This inflammatory process led to permanent neuroglia changes and further retinal degeneration. Infiltration of blood derived monocytes in the retina was shown to be specific to retinal injury, and no physiological turnover of microglia was present in normal eyes.

CONCLUSIONS: This study provides a plausible mechanism to explain the otherwise enigmatic, often severe, glaucoma in the absence of intraocular pressure elevation. In addition, this study demonstrated the differential and detrimental role of blood-derived monocytes in neuroglia changes following corneal injury. Thus, based on the convergence of clinical and animal studies, treatment might included the following three steps: 1. Prompt systemic inhibition of TNF-α (infliximab 5-10 mg/kg), in addition to lavage.

2. L ong-term prophylactic intraocular pressure reduction to the lowest safe level with carbonic anhydrase inhibitors.

3. Keratoprosthesis surgery in later quiet stage, if indicated.

With such a “triple” approach, prognosis for even severe burns will likely improve. These principles might also be applicable after any corneal surgery that triggers substantial inflammation.

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30th Biennial Cornea Conference

Primary Cilium Disruption is Associated with Anterior Segment Dysgenesis Celine Portal1,*, Qing Liu1, Carlo Iomini1

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Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY.

1

*Email: celine.portal@mssm.edu PURPOSE: Anterior segment dysgenesis (ASD) represents one of the main causes of congenital opacity, and leads to glaucoma in ~50% of the cases. Several ciliopathies including Meckel, and Joubert syndromes display conditions affecting tissues of the anterior segment (AS). However, the role of cilia during AS development is poorly understood.

METHODS: To investigate the role of cilia in AS development, we used Ift88-/- mice and a conditional knock-out mouse Wnt1-Cre;Ift88fx/- (cKO). To monitor the Cre activity, the Rosa26mT/mG reporter line (mT/mG) was used in the cross Wnt1-Cre;Ift88fx/fx; mT/mG. Prior to Cre-mediated excision, mT/mG mouse express a red-fluorescent membrane-targeted tdTomato (mT). In presence of a Cre, the excision of the mT cassette allows the expression of a membranetargeted green fluorescent protein (mG). cKO mice were also crossed with the Gli1LacZ mouse to assess Hedgehog (Hh) signaling activation. Phenotypic analysis of the anterior segment (AS) in null and conditional ciliary mutants was performed using microscopy. Cell proliferation of POM cells was assessed by bromodeoxyuridine labeling.

RESULTS: Null mutations that prevent cilia assembly such as Ift88-/- are lethal at E9.5-10.5. In Ift88-/- embryos the optic vesicle, although defective, is present. However, the optic cup does not form at E10.5, as observed in wild-type embryos. To overcome mid-gestation lethality, we generated a cKO mouse Wnt1-Cre;Ift88fx/- in which the Ift88 gene is excised in periocular mesenchyme (POM) cells of neural crest origin. cKO mice display strong craniofacial defects and die at birth. In late stages of embryonic development, cKO mice display significant reduction of the anterior chamber, a thinner stromal extracellular matrix and corneal endothelial malformations.

Interestingly, although migration of neural crest-derived cells appears normal, we detected reduction of mesenchymal cells at the angle between the stroma and the optic cup at E17.5. At E18.5, stromal keratinocytes exibit an increased number of filopodia like filaments. Moreover, we found a dramatic disruption of the Hh signaling and cell proliferation in a subpopulation of POM cells surrounding the optic nerve and the retinal pigmented epithelium up to the iridocorneal angle of cKO mice.

CONCLUSIONS: Primary cilia play a pivotal role in AS development and are involved in the etiology of ocular disorders derived from ASD.

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Harvard Department of Ophthalmology

47

Activation of NLRP3 Pathway is Required for clearance P. aeruginosa Infection in Mouse Corneas Abdulraouf Ramadan1, Zhiyi Cao1, Vijay A. Rathinam2, Mihaela Gadjeva3, Noorjahan Panjwani1

New England Eye Center/Department of Ophthalmology, Tufts University School of Medicine, Boston, MA; 2Department of Immunology, UConn Health School of Medicine, Farmington, CT; 3 Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 1

RATIONALE: The role of inflammasome pathway in the regulation of P. aeruginosa (PA) keratitis is unclear. The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance PA infection in mouse corneas.

METHODS: Corneas of C57BL/6 mice were infected with PA (strain 6077). Immediately prior to infection, animals received subconjunctival injection of the NLRP3 inhibitor (300µM in 10 µl PBS) or PBS. The severity of bacterial keratitis was graded on day 1 and day 3 post-infection (pi) by slit lamp using a scoring system ranging from 0 to 4 as described, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) analysis of cleaved caspase-1 and IL1-β by Western blot analysis, and (iii) IL1β quantification by ELISA. RESULTS: Mice receiving subconjunctival injections of NLRP3 inhibitor exhibited more severe infection as indicated by increase in opacity score and increased bacterial load. The hallmark of inflammasome activation is the activation of proinflammatory caspase-1 by cleavage of its precursor, procaspase-1; active (cleaved) caspase-1 subsequently cleaves pro-IL-1β to generate mature/cleaved IL1β which is secreted from the cell to mediate downstream inflammatory effects that clear the infection. Accordingly, we found that NLRP3 inhibition resulted in reduced recruitment of immune cells (CD45+ cells, neutrophils [CD45+CD11b+LyG6+], and macrophages [CD45+CD11b+F4/80+]), reduced levels of mature/cleaved forms of caspase-1 and IL1β in infected corneas. Additional pilot studies revealed that NLRP3 pathway is also activated in human corneal epithelial cells infected with PA. CONCLUSIONS: Activation of NLRP3 pathway is required for clearance of PA infection in mouse corneas. Corneal epithelial cells as well as immune cells may play a role in the activation of NLRP3 pathway in corneas infected with P. aeruginosa.

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30th Biennial Cornea Conference

Morphological alterations following MGAT5 abrogation in human corneal epithelial cells Maria C. Rodriguez Benavente1,*, Takazumi Taniguchi1, Pablo ArgĂźeso1

48

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

*Email: maria_rodriguezbenavente@meei.harvard.edu PURPOSE: Golgi β1,6-N-acetylglucosaminyltransferase V (MGAT5) produces complex branched N-glycans on glycoproteins implicated in cell processes, such as cell differentiation and senescence. Here, we investigated the effect of in vitro abrogation of MGAT5 in order to dissect the contribution of this pathway to ocular surface homeostasis.

METHODS: Immortalized human corneal epithelial cells were grown to confluence in supplemented keratinocyte serum-free medium, and induced to differentiate into a stratified epithelium using serum-containing medium. Depletion of MGAT5 was achieved using a double-hit siRNA transfection strategy. Total RNA was isolated and reverse-transcribed into cDNA in order to determine the relative levels of MGAT5 gene expression by qPCR. Cell morphology was assessed by phase-contrast microscopy. Barrier function was evaluated using a rose bengal assay.

RESULTS: As shown by qPCR, abrogation by siRNA in stratified corneal epithelial cells resulted in an 80% reduction of MGAT5 expression levels compared to scrambled siRNA control. Phasecontrast microscopy revealed abnormal phenotypic changes in the MGAT5-abrogated corneal epithelial cells, which acquired a spindle shape morphology and showed noticeable loss of cellcell contacts. Furthermore, MGAT5 abrogation produced loss of barrier function, as shown by the reduction in the number of islands excluding the rose bengal dye.

CONCLUSIONS: Taken together, this study suggests that MGAT5 knockdown alters the morphology and barrier function of corneal epithelial cells.

SUPPORT: NIH Grant R01EY026147

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Harvard Department of Ophthalmology

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Contribution of ABCB5-positive Limbal Stem Cells to Corneal Epithelial Development and Regeneration Yuzuru Sasamoto1,*, Gabriel Gonzalez1,2, Bruce R. Ksander3, Markus H. Frank4,5, Natasha Y. Frank1,2,3.

Division of Genetics, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA; Department of Medicine, VA Boston Healthcare System, Boston, MA; 3Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; 4Transplant Research Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA; 5Harvard Stem Cell Institute, Harvard University, Cambridge, MA 1 2

*Email: ysasamoto@bwh.harvard.edu PURPOSE: Our laboratories demonstrated that ATP-binding cassette (ABC) superfamily member B5 (ABCB5) identifies limbal stem cells (LSCs) with the ability to restore and maintain the corneal epithelium upon transplantation in preclinical models of limbal stem cell deficiency (LSCD). In the current study we employed a novel genetic lineage tracing model to dissect the developmental origin of ABCB5+ LSCs and the developmental cell fates of their progeny.

METHODS: Abcb5/Cre transgenic mice were generated by insertion of an IRES-Cre cassette in the Abcb5 3’UTR downstream of the STOP codon located in exon 30. Abcb5/Cre mice were crossed with tdTomato (B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) mice for genetic lineage tracing studies.

RESULTS: Whole-mount cornea preparations from Abcb5/Cre/tdTomato mice allowed identification of tdTomato-positive Abcb5-derived progeny cells within the entire adult mouse corneal epithelium. Immunofluorescent images revealed that tdTomato and Abcb5 were coexpressed in the limbus of Abcb5/Cre/tdTomato mice.

CONCLUSIONS: We have identified at the level of genetic lineage tracing that, consistent with their LSC phenotype, ABCB5-expressing precursor cells give rise to the entire corneal epithelium during development and regeneration.

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Characterizing the Ciliary Nerve Ligation Model of Corneal Neuropathic Pain Yashar Seyed-Razavi1,*, Maria J. Lopez1, Takefumi Yamaguchi2, Pedram Hamrah1,3

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Center for Translational Ocular Immunology, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine Boston, MA; 2Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan; 3 Cornea Service, New England Eye Center, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 1

*Email: yseyedrazavi@tuftsmedicalcenter.org PURPOSE: The purpose of this study was to develop and characterize a murine model of corneal neuropathic pain.

METHODS: Following a lateral canthotomy, two tractional 5-0 nylon sutures were placed temporally and the lateral conjunctival fornix incised circumferentially (90 degrees) taking care not to damage the retro-orbital venous plexus. The eye globe was rotated nasally and a 7-0 silk suture was placed and tightened around the exposed optic nerve and surrounding ciliary nerve branches, ligating the ciliary nerves without severing them. Following the procedure, 8–0 nylon sutures for tarsorrhaphy were placed. Mice undergoing all surgical steps yet lacking the ligation suture served as sham controls. An eye-wiping test was used to evaluate corneal sensitivity at baseline and 3, 7, and 14 days post surgery (dps) with 10μL of hyperosmolar saline solution [2M] applied to treated eyes. Light and fluorescein stained images of the ocular surface were also acquired with a slitlamp. Excised corneal flat-mounts were stained with anti-βIII tubulin to assess nerve alterations.

RESULTS: Minimal corneal staining was observed in sham and ligation groups at days 3 (p=0.32), 7 (p=0.37) and 14 (p=0.67). Analysis of the eyelids and area surrounding the eye revealed blepharitis and hair loss compared to sham controls (p<0.01). Corneal sensation was retained post ligation while hypersensitivity to hyperosmolar saline solution was noted at all time-points when compared to sham controls, and to baseline in naïve mice (p<0.001, one-way ANOVA). Analysis of corneal flat-mounts revealed that ligation did not result in nerve avulsion; rather, anatomical and density alterations (beading and neuromas) were noted in the subbasal and stromal corneal nerves.

Nerve density was altered in the central subbasal plexus 14 dps (p<0.05) but not in the periphery.

CONCLUSIONS: We have developed a minimally invasive, simple and novel murine model of corneal neuropathic pain by way of ligation of ciliary nerves. This model can be applied towards understanding ocular pain, investigating the underlying mechanisms of nerve function in a neuropathic setting, as well as the therapeutic effect of drugs in the treatment of this debilitating disease.

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Harvard Department of Ophthalmology

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Keratolimbal Allograft for Limbal Stem Cell Deficiency after Severe Corneal Chemical Burn: A Systematic Review Swapna S. Shanbhag1,*, Hajirah N. Saeed1, Eleftherios I. Paschalis1, James Chodosh1

Disruptive Technology Laboratory, Mass. Eye and Ear, Boston, MA

1

*Email: swapna_shanbhag@meei.harvard.edu PURPOSE: To review the published literature on outcomes of keratolimbal allograft (KLAL) for the surgical treatment of limbal stem cell deficiency (LSCD) and corneal blindness after severe corneal chemical burn.

METHODS: Literature searches were conducted in the following electronic databases: MEDLINE, EMBASE, Science citation index, CINAHL, LILACS, and the Cochrane Library. Standard systematic review methodology was applied. Reference lists were scanned to identify additional applicable studies. The main outcome measure was the proportion of eyes with best-corrected visual acuity (BCVA) ≥ 20/200 at last follow-up. Other measures of allograft success were also collected.

RESULTS: We identified 6 reports in which KLAL outcomes in eyes with chemical burns could be distinguished. There were no randomized controlled studies. All KLAL recipients received oral immunosuppression. The outcomes of KLAL in 36 eyes of 33 patients were analyzed. One study with 7 eyes did not specify KLAL follow-up specific to chemical injury. Median postoperative followup for the other 29 eyes in 26 patients was 42 months (range 6.2-114 months). In the same 29 eyes, 69% (20/29) had BCVA ≥ 20/200 at the last follow-up examination. Eighty nine percent of all eyes (32/36) underwent penetrating keratoplasty simultaneous or subsequent to KLAL. Rejection of the central corneal graft occurred in 37.5% (12/32). Additional surgical interventions were required in 61.1% (22/36) eyes.

CONCLUSIONS: The number of studies where outcomes of KLAL in eyes with severe corneal chemical burns could be discerned was limited, and large variability was observed in outcome reporting. The overall quality of evidence to support the use of KLAL in LSCD in severe chemical corneal burns was low. Standardization and longer follow-up are needed to better define evidencebased best practice when contemplating surgical intervention for corneal blindness after severe corneal chemical burn.

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Diphtheroids as a Corneal Pathogen in Chronic Ocular Surface Disease Grace Shih1,*, Deborah Jacobs1, Hajirah Saeed1

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Harvard Ophthalmology/Mass. Eye and Ear

1

*Email: grace_shih@meei.harvard.edu PURPOSE: To characterize the presentation and natural history of diphtheroid keratitis.

METHODS: Retrospective medical record review.

RESULTS: Four cases of culture proven diphtheroid keratitis were identified: Case 1: 59-year-old male with acute Stevens-Johnson syndrome (SJS) 5 years earlier, with persistent epithelial defect that progressed to melting and perforation. Case 2: 70-year-old male with acute SJS 6 months earlier and persistent epithelial defect with thinning. Case 3: 42-year-old female with history of SJS over 20 years earlier, with epithelial defect, thinning, and focal opacity in the right eye (R), and 8 months later, epithelial defect and hypopyon in the left eye (L). Clinical findings and images were notable for lack of infiltrate typically associated with microbial infection. Diphtheroids were identified as the predominant organism in the initial culture of all 4 cases of keratitis. Speciation revealed Case 1: Corynebacterium glucuronolyticum, Case 2: Corynebacterium amycolatum, Case 3R: Corynebacterium striatum, and Case 3L: Corynebacterium amycolatum, No sensitivities were recorded for the first case. All other cases were sensitive to vancomycin, and Case 2 and 3L were also sensitive to clindamycin and tetracycline. Case 3L was additionally sensitive to penicillin.

Case 1 was stabilized with penetrating keratoplasty, amniotic membrane grafting, tarsorraphy, and postoperative moxifloxacin. The latter three cases healed with arrest of thinning on topical vancomycin. The focal opacity in Case 3R eye cleared over months. Retrospective review reveals associated risk factors of topical steroid use, SJS with keratinization, and PROSE treatment with overnight wear in all cases, as well as recent therapeutic soft lens (Cases 1, 2), prophylactic antibiotic use (Case1, 2, 3L: moxifloxacin; Case 1: gatifloxacin; Case 3R: ciprofloxacin), and systemic immune compromise (Case 1: Type II DM; Case 3: treatment of secondary chondrosarcoma of lung).

CONCLUSIONS: In contrast with other gram positive bacteria, diphtheroid corneal infections present in more indolent fashion. These cases exhibited persistent epithelial defects with gradual thinning and minimal injection, discharge, opacity or infiltrate. Clinicians should consider culture of a persistent epithelial defect in patients with multiple risk factors for infection, even in the absence of infiltration. Diphtheroids should be treated if they are identified as the dominant organism.

Recognition and treatment of Corynebacteria spp. as opportunistic pathogens can lead to favorable outcomes in cases of seemingly sterile ulceration during the chronic phase of StevensJohnson syndrome.

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Harvard Department of Ophthalmology

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Epidemiological Aspects and Predisposing Factors of Microbial Keratitis Delta Region of Egypt Doaa H. Sobeih1,*, Nashwa S. Sadik1

Mansoura Ophthalmic Hospital, Mansoura, Egypt

1

*Email: doaa_sobeh83@yahoo.com PURPOSE: The aim of the current study is to investigate epidemiological characteristics of the patients who were diagnosed with microbial keratitis.

METHODS: Patients who were diagnosed with microbial keratitis were included in the study.

Demographic characteristics like age, sex, occupation, place of residence were collected and analyzed. We have identified those who had predisposing factors like contact lens wear, systemic diseases, trauma, and ocular diseases.

RESULTS: A total of 300 patients who were diagnosed with microbial keratitis with a mean age of 56Âą8 years (177 male and 123 females). Of those, 78% had agricultural activities and live in rural areas in Delta region of Egypt. The majority of the patients showed irrelevant predisposing factors or nearly minor trauma. Corneal swaps from 246 patients (82%) grew positive cultures. Of those, surprisingly, nearly 80% grew positive cultures for fungal isolates. Gram positive and gram negative bacterial isolates accounts for 14% and 6%, respectively. Staph aureus was the predominant gram positive bacteria while pseudomonas aeruginosa was the predominant gram negative organism detected in cultures. Other predisposing factors like contact lens wearing, dry eye, and abuse of topical eye drops were detected in a small percent of cultures.

CONCLUSIONS: Fungal keratitis was reported in the majority the culture proven cases presented in rural areas of Egyptian Delta. This might be attributed to factors like hot and humid environment alongside with lack of awareness about healthy habits.

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AAV Gene Delivery via Subconjunctival Injection in Mice Liujiang Song1,2,3, Laura Conatser1,2, Llanga Telmo1,2, Brian C. Gilger4, Matthew Hirsch1,2

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Ophthalmology, University of North Carolina, Chapel Hill, NC; 2 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 3Department of Pediatrics, Hunan Normal University Medical College, Changsha, Hunan, China; 4College of Veterinary Medicine, North Carolina State University, Raleigh, NC 1

PURPOSE: AAV based gene therapy approaches have demonstrated promising clinical results for the treatment of posterior eye disorders, however, application of this technology to anterior eye disorders has been relatively unexplored in preclinical applications. In particular, a comprehensive report of this technology in subconjunctival injections is lacking. To determine the validity of this route and optimal AAV serotype for anterior eye gene therapy, a survey of natural and mutant AAV serotypes was performed in a wt murine model. METHODS: Pseudoserotypes harbouring the ITR of AAV2 and the capsids from different serotypes carrying an EGFP reporter gene under a CMV promotor was administered subconjunctivally. Following AAV vector administration, the serum was collected over a 2 month period, hepatic damage was assessed by detecting Aspartate aminotransferase and Alanine aminotransferase in serum. The ocular surface was evaluated by direct observation and a slit lamp to assess any ocular disturbances such as eye redness, cornea opacity, surface smoothness, conjunctival congestion, swelling and discharge. Tear production was measured by a ZoneQuick phenol red thread test. The neutralization assay was conducted to determine the antiAAV antibody. The vector shedding was assessed qPCR. Post-mortem analyses included GFP abundance in distinct anterior eye compartments, cell type specific transduction.

RESULTS: Histological analysis showed the transgene expression is serotype dependent, qPCR analysis demonstrated different amounts of viral genomes in different ocular tissues.

Subconjunctivally delivered AAV vectors did not elicit any severe hepatic injury or systemic immunogenicity.

CONCLUSIONS: Cell type/tissue specific transcripts/transgene abundance is dependent on vector capsid serotype. Subconjunctivally administered AAV may be a promising gene delivery approach for managing anterior segment eye diseases such as dry eye disease.

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Harvard Department of Ophthalmology

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Ocular Pain in Patients with Dry Eye Disease Zhongmou Sun1, Vannarut Satitpitakul2, Ahmad Kheirkhah1,2, Alja Crnej2, Pedram Hamrah3, Reza Dana1,2

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; 3Cornea Service, New England Eye Center, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 1 2

PURPOSE: To evaluate the level of ocular pain in patients with dry eye disease (DED) and to correlate the ocular pain with the Ocular Surface Disease Index (OSDI) score and DED signs.

METHODS: This cross-sectional study included 91 patients with DED. All patients were asked to score their level of ocular pain using a 10-point scale, with 10 indicating the most severe ocular pain. All patients also had a comprehensive ophthalmic assessment including OSDI questionnaire.

The correlations between the level of ocular pain and OSDI and clinical parameters were then evaluated.

RESULTS: 85.4% of patients with DED had some degree of ocular pain, with mild pain (scores <5) in 44.9%, moderate pain (scores 5-7) in 33.7% and severe pain (scores >7) in 6.8%. The mean level of ocular pain was 4.1 Âą 2.4 with a median of 3. The mean OSDI score was 44.7 Âą 22.7. The level of ocular pain had a statistically significant correlation the OSDI score (P=0.01) though with a relatively low coefficient (Rs=0.32). No correlations were found between the level of ocular pain and DED signs.

CONCLUSIONS: Majority of patients with DED have ocular pain which correlates only weakly with the OSDI score. Evaluation of the level of ocular pain should be considered in routine assessment of patients with DED.

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Safety and Efficacy of Wiping the Lid Margins with Lid Hygiene Shampoo Using “Eye Brush,” A Lid Hygiene Brush Prototype, In Normal Subjects: A Pilot Study

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Hirotaka Tanabe1,2, Motoko Kawashima2, Minako Kaido2, Reiko Ishida2, Tetsuya Kawakita2, Kazuo Tsubota2 Ophthalmology, Ashikaga Red Cross Hospital, Ashikaga, Tochigi, Japan; 2Ophthalmology, Keio University School of Medicine, Shinjuku, Tokyo, Japan 1

COMMERCIAL RELATIONSHIPS DISCLOSURE: Hirotaka Tanabe: Commercial Relationship(s); Eye Brush (Application number 2015-024983, Japan) / Keio University School of Medicine: Code P (Patent) | Motoko Kawashima: Commercial Relationship(s); MediProduct Co., Ltd. (Eye Shampoo): Code F (Financial Support) | Minako Kaido: Commercial Relationship: Code N (No Commercial Relationship) | Reiko Ishida: Commercial Relationship: Code N (No Commercial Relationship) | Tetsuya Kawakita: Commercial Relationship: Code N (No Commercial Relationship) | Kazuo Tsubota: Commercial Relationship(s); Eye Brush (Application number 2015-024983, Japan) / Keio University School of Medicine: Code P (Patent); MediProduct Co., Ltd. (Eye Shampoo): Code F (Financial Support) Study Group: (none) PURPOSE: We performed a pilot study to evaluate the safety and efficacy of using a lid hygiene brush prototype (Eye Brush) to wipe the lid margins with lid hygiene shampoo (Eye Shampoo) in normal subjects.

METHODS: Twelve eyes of 6 subjects (6 males aged 32-56 [39.8±9.62] years) with normal meibomian glands were evaluated before and after wiping the lid margins in four ways: with tap water alone or using Eye Brush, Eye Shampoo (MediProduct, Japan) or both. Each procedure was performed during a different week. The results after using both Eye Brush and Eye Shampoo 2 times daily for 1 month were also evaluated. The efficacy of each method was determined based on the washout rate of fluorescein-stained 0.3% Tarivid ophthalmic ointment, which was fully applied to the eyelids, including the lid margins. We used Wilcoxon signed-rank tests to analyze the tear break-up time (TBUT) (grades 0-10), corneal and conjunctival fluorescein/lissamine green/ rose bengal staining scores (grades 0-9), lid margin lissamine green staining scores (grades 0-3), subjective symptoms (grades 0-100, as assessed via a visual analog scale [VAS]), tear lipid layer interference (grades 1-5, as assessed via a DR-1 tear interference camera [Kowa Co., Nagoya, Japan]), and post-wiping scores for remaining ointment on the eyelids (grades 0-6).

RESULTS: Deterioration (P<0.05) was not observed after any method in terms of the TBUT, corneal and conjunctival staining scores or lid margin staining scores. Regarding the VAS scores, dryness was significantly improved with Eye Brush (13.3±12.0 to 2.16±3.43, P=0.014), and eye discharge significantly increased after 1 month of using both Eye Brush and Eye Shampoo (2.08±3.11 to 5.00±4.09, P=0.042). The wiping efficacy when using Eye Brush, Eye Shampoo or both was significantly greater than when using tap water alone (two-sided test, P=0.003, 0.003, 0.002), and using both was significantly more efficient than using Eye Shampoo alone (one-sided test, P=0.009).

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Harvard Department of Ophthalmology

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High-Intensity Accelerated Corneal Cross-Linking as a Therapy for Keratitis: Chromophore-Photoactivation with Low Levels Of Hydrogen Peroxide (Bperox) in vitro Andreina Tarff1, Rebecca Yee2, Ying Zhang2, Ashley Behrens1

The Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine; 2Molecular Microbiology & Immunology, Johns Hopkins University, Baltimore, MD 1

PURPOSE: Accelerated methods for corneal cross-linking (CXL) have been introduced clinically as an adjuvant therapy for keratitis. CXL oxygen dependency stills a concern in the effectiveness of such expedited methods, and hydrogen peroxide (H2O2) is a well-known therapeutic oxygen carrier compound. Therefore, we aim to potentiate its bactericidal activity by adding a rich oxygen environment from low levels of H2O2.

METHODS: Multidrug resistant-moxifloxacin resistant Staphylococcus aureus (MDR-MXRSA), carbapenemase producing Klebsiella pneumonia (KP-C), multidrug resistant Pseudomonas aeruginosa (MDR-PA) and multidrug resistant-fluoroquinolones family resistant Acinetobacter baumannii (MDR-FRAB) (107 CFU/ml) were exposed in 12-well culture plates to BPerox, which contains riboflavin (RF) 0.5% plus H2O2 0.004%, photoactivated by two different UVA doseregimens: A) ultra-rapid, 30 mW/cm2 for 3 min and B) rapid, 10 mW/cm2 for 10 min, at a wavelength of 365 nm through an 8-mm iris aperture. Control groups included alone treatments with BPerox, RF, UVA and H2O2, combination treatments with RF+UVA and H2O2+UVA, and the untreated sample. Cell viability was assessed after 24 hours of incubation at 37 °C by cell forming unit (CFU) enumeration. Triplicate values were obtained. Student’s t-test was used for statistical analysis.

RESULTS: MDR-MXRSA and KP-C showed complete sterilization (0 CFU) after treatment with BPerox (B2 0.5% + H2O2 0.004%) photoactivated by UVA regimens A and B. MDR-PA and MDRFRAB also had complete bactericidal activity (0 CFU) after treatment with BPerox photoactivated

by UVA regimen B (p ≤ 0.0008), and for regimen A the decreased in the bacterial load was statistically significant (p ≤ 0.0272), compared to all controls that resulted with at least 106 CFU/ml cells remaining, demonstrating none microbicidal activity.

CONCLUSIONS: High irradiance accelerated CXL methods using RF-photoactivation with low levels of H2O2 are able to achieve total killing at treatment exposure times as short as 3 min. Our new therapy may help to restore corneal lamella strengthening, more than conventional CXL.

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The Impact of Corneal Topography and Biomechanics on Refractive Error in Young Healthy Subjects Mengyu Wang1, Yaohua Zhang2,3, Wenjing Wu2,3, Tobias Elze1,Yan Wang2,3,*

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Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Harvard Boston, MA; 2Clinical College of Ophthalmology, Tianjin Medical University, Tianjin, China; 3Tianjin Eye Hospital & Eye Institute, Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin, China.

1

*Email: wangyan7143@vip.sina.com PURPOSE: Previous research has shown the corneal topographic and biomechanical properties (CTBPs) measured by Ocular Response Analyser to be related to refractive error in healthy subjects, while it is not clear whether the effect of biomechanical properties on refractive error is independent of various known topographic features. In this study, using biomechanical properties measured by new noncontact tonometry (Corvis ST), we systematically study the relationship between a large number of CTBPs and refractive error.

METHODS: For each subjects, we randomly select one eye per subject. For each eye, CTBPs measured by Pentacam and OCULUS Corvis ST were extracted from the machines. Multivariate linear regressions were performed to associate CTBPs in addition to gender and age with spherical equivalent (SE), sphere and cylinder, respectively. As many of the CTBPs are inter-correlated, model selection based on Bayesian information criterion (BIC) was performed to select the optimal model to predict SE, sphere and cylinder, respectively.

RESULTS: 280 eyes from 280 subjects were used in this study with 173 male and 107 female.

The mean ± standard deviation of age and spherical equivalent (SE) were 21.8±5.3 years and -5.3±2.0 diopters. Table 1 shows that posterior astigmatism, gender, flattest anterior curvature (FAC), first applanation (A1) velocity and second applanation (A2) time were positively significantly associated with SE, while steepest anterior curvature (SAC), corneal central thickness (CCT) and highest concavity deformation amplitude (HCDA) were inversely significantly associated with SE. Female was more myopic than male. The optimal subset of parameters to predict SE included positively associated posterior astigmatism, FAC, intraocular pressure (IOP), A1 velocity, and A2 time, and negatively associated SAC, CCT and HCDA. The optimal subset of parameters to predict sphere included positively associated IOP, A1 velocity and A2 time, and negatively associated SAC, CCT and HCDA. The optimal subset of parameters to predict cylinder included positively associated posterior astigmatism, FAC, A2 length, A2 velocity and HCDA, and negatively associated SAC. CONCLUSIONS: We demonstrate that the CTBP parameters are substantially associated with refractive error. Our findings might help to identify the CTBP risk factors that affect the outcome of refractive error surgery.

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Harvard Department of Ophthalmology

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Prosthetic Replacement of the Ocular Surface Ecosystem for Ocular Surface Disease in Pediatric Patients with Stevens-Johnson Syndrome Yvonne Wang1,*, Hajirah N. Saeed1, Rohini Rao1, Deborah S. Jacobs1,2

Harvard Ophthalmology/Mass. Eye and Ear, Boston, MA; 2BostonSight, Needham, MA

1

*Email: Yvonne_wang@meei.harvard.edu PURPOSE: Prosthetic replacement of the ocular surface ecosystem (PROSE) treatment uses customized scleral lens prosthetic devices to support ocular surface function in ocular surface disease (OSD). We present an outcomes study of PROSE treatment in the pediatric patients affected by Steven-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN).

METHODS: This is a retrospective cohort study. We reviewed records from 1997 through 2016 from the Boston Foundation for Sight, of patients age 18 or younger with SJS/TEN, who were seen in consultation for PROSE treatment.

RESULTS: 44 pediatric patients were seen in consultation. 69 eyes were fitted for PROSE and successful wear was documented in 58 eyes. The most common visual symptom at the time of presentation was photophobia. The median age of SJS onset was 6.7 years (range 2-16). The median age of initial presentation to BFS was 8.1 years (range 4-17). The median age at which PROSE wear started was 9.2 years (range 4-16). Fitting and training (F&T) with the PROSE device occurred in an average of 3.9 months. Eight out of 43 patients failed F&T, and the average age of those who failed was 11 (range 4-17). Median visual acuity at initial visit was 0.60 (Snellen equivalent of 20/80). The median visual acuity with initial PROSE wear was 0.18 (Snellen 20/30, p<0.002). Median visual acuity with PROSE at last follow up was 0.18 (Snellen 20/30, p<0.002) over a mean follow up time of 6.1 years. Patients had an average wear time of 13 hours a day. The average lens diameter at the final fitting was 17.5 mm.

CONCLUSIONS: PROSE treatment is feasible in pediatric patients with OSD caused by SJS/TEN, with improvement and stabilization of vision similar to what has been reported for adults. 80% of patients were able to successfully wear the PROSE device, even those as young as age 4.

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Menadione-Induced Endothelial-Mesenchymal Transition in Human Corneal Endothelial Cells - A Cell Model Tomas L. White1,*, Kishore Reddy Katikireddy1, Shivakumar Vasanth1, Taiga Miyajima1, Duna Raoof1, Yuming Chen1, Marianne Price3, Francis W. Price3, Ula V. Jurkunas1

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Schepens Eye Research Institute of Mass. Eye and Ear, Boston, MA; 2AbbVie Deutschland GmbH & Co KG, Knollstraße, Ludwigshafen, Germany; Cornea Research Foundation of America, Indianapolis, IN 1

*Email: Tomas_White@meei.harvard.edu PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is a genetic, oxidative stress disorder of post-mitotic corneal endothelial cells (CEnCs), which normally exhibit hexagonal shape and form a compact monolayer compatible with normal corneal functioning and clear vision. FECD is associated with increased DNA damage, which in turn leads to CEnC loss forming rosettes and aberrant extracellular matrix (ECM) deposition in the form of pro-fibrotic guttae. Since the mechanism of ECM deposition in FECD is currently unknown, we aimed to investigate the role of endothelial-mesenchymal transition (EMT) in FECD using a previously established cellular in vitro model that recapitulates the characteristic rosette formation.

METHODS: CEnC’s were subjected to 25 and 50μM menadione (MN) to induce oxidative stress.

Additionally, 10ng/ml TGFβ1 was used alone or in combination with MN. Morphological changes were detected with phase-contrast microscopy, and cell viability was assessed with tryptan blue dye exclusion assay. EMT markers were examined using RT-PCR, western blot, and immunofluorescence microscopy.

RESULTS: We demonstrate that MN treatment decreased cell viability and increased intracellular ROS levels in CenC’s during rosette formation; this effect was potentiated with the addition of TGFβ1. Additionally, MN upregulated EMT- and FECD-associated markers such as Snail1, fibronectin, N-cadherin, ZEB1 and TGFBI, clusterin, respectively. Furthermore, an upregulation of EMT was observed in FECD ex vivo specimens, with a loss of organized junctional staining of plasma membrane-bound N-cadherin and increase in fibronectin and Snail1 compared to ex vivo controls. Addition of N-acetylcysteine (NAC) downregulated all EMT markers and abolished rosette formation. Loss of NQO1, a metabolizing enzyme of MN, led to greater increase in ROS, apoptosis and upregulation of Snail1, fibronectin, and N-cadherin than in normal cells, indicating that NQO1 regulates Snail1-mediated EMT. CONCLUSIONS: This study provides first line of evidence that MN-induced oxidative stress leads to EMT in corneal endothelial cells, the effect of which is further potentiated when redox cycling activity of MN is enhanced by the absence of NQO1.

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Harvard Department of Ophthalmology

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Inflammatory Stress Impairs the N-Glycan Branching Pathway in Human Ocular Surface Epithelia Ashley M. Woodward1,*, Sylvain Lehoux2, Flavio Mantelli3, Stefano Bonini4, Pablo Argüeso1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 3Dompé farmaceutici S.p.A., Milan, Italy; 4University Campus Bio-Medico, Rome, Italy 1 2

*Email: Ashley_Woodward@meei.harvard.edu PURPOSE: Regulation of the medial Golgi N-glycan branching pathway is critical for cell growth, arrest, and differentiation. Here, we investigated the effect of inflammation on N-glycosyltransferase expression at the ocular surface.

METHODS: N-glycans isolated from stratified human corneal-limbal epithelial cells were profiled

by MALDI-TOF mass spectrometry. The effect of inflammatory mediators on the expression of N-glycosyltransferases was analyzed using a human glycosylation PCR array and qRT-PCR. Conjunctival biopsies and impression cytology samples were collected from healthy subjects and patients with ocular cicatricial pemphigoid.

RESULTS: By mass spectrometry, corneal epithelial cells were rich in high mannose-type structures and non-bisected biantennary complex-type N-glycans. Treatment with TNFα resulted in an early endoplasmic reticulum stress response, followed by a down-regulation of 17 N-glycosyltransferases. Of these enzymes, six were involved in the medial Golgi N-glycan branching pathway and included MGAT1 and 2, which are required for the formation of biantennary structures. Compared to TNFα, IL-1β had a limited effect on gene expression that included the bisecting medial Golgi MGAT3. Analysis of ocular pemphigoid tissue revealed elevated endoplasmic reticulum stress and a concomitant decrease in MGAT1 and 2 compared to controls.

CONCLUSIONS: This study demonstrates that pro-inflammatory conditions lead to alteration of the N-glycan branching pathway in the medial Golgi, which may contribute to the pathophysiology of ocular surface disease.

FUNDING: NIH Grant EY026147

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Outcomes of Double-Headed Pterygium Surgery Performed with Double Conjunctival Autografts Jeannie Xu1, Hyunjoo J. Lee1

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Ophthalmology, Boston University School of Medicine, Boston, MA

1

PURPOSE: Double-headed pterygium, the presence of both nasal and temporal pterygia in the same eye, are often treated with a single conjunctival autograft (CAG) and a contralateral amniotic membrane transplantation (AMT), perhaps due to concern about the safety and feasibility of harvesting two CAG from the same eye. We aimed to examine the safety and efficacy of performing double-headed pterygium excision with double CAG by reviewing our cases, and to compare these outcomes to cases performed with single CAG and contralateral AMT. METHODS: A retrospective chart review of double pterygium surgery cases at Boston Medical Center performed between September 2013 and December 2016 by a single surgeon. Outcome measures included: rate of pterygium recurrence, pre-operative and post-operative BCVA, and complications.

RESULTS: 10 eyes of 8 patients who underwent double pterygium surgery were identified. 6 eyes received double CAG alone, and 2 eyes received double CAG to cover the limbal defects, supplemented with AMT to cover any remaining posterior conjunctival defect at the excision sites. 2 eyes of the same patient received a single CAG, and AMT was used to cover the entire contralateral defect, due to concern about mild glaucoma suspicion. None of the surgeries involved mitomycin-C application. No recurrences were noted in the 8 eyes in which double CAG ± supplemental AMT was performed (0 of 8 eyes, follow-up 0.25-12.25 mo, avg 3.7 mo). In eyes with single CAG and contralateral AMT, 1 pterygium recurrence occurred at the site of a temporal AMT at 5 months (1 of 2 eyes, follow-up 12.5-16 mo, avg 14.3 mo). The average pre-operative and final post-operative logMAR BCVA was 0.29 ± 0.33 SD (Snellen equivalent 20/39) and 0.10 ± 0.16 SD (20/25) in the double CAG group and 0.05 ± 0.07 SD (20/22) and 0.05 ± 0.07 SD (20/22) in the single CAG+AMT group, respectively. No complications were identified with performing either a double CAG or single CAG+AMT. In particular, no limbal stem cell deficiencies or complications of graft harvest site healing were noted with double CAG. CONCLUSIONS: The use of double CAG to cover all limbal defects in cases of double pterygium excision was not associated with any complications, and was associated with a low pterygium recurrence rate. The use of double CAG or single CAG+AMT for double pterygium excision was associated with good visual outcomes.

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Ethnic and Racial Differences in Prevalence of Meibomian Gland Dysfunction within the Older Population Siri P. Yalamanchili1,*, Jared Donaldson2, Michael F. Ward, II2, Richard Davis2

Northeast Ohio Medical University, Rootstown, OH; 2Department of Ophthalmology, University of North Carolina, Chapel Hill, NC 1

*Email: syalamanchili@neomed.edu PURPOSE: Meibomian gland dysfunction (MGD) is the principle cause of evaporative dry eye disease (DED), and is a main cause of discomfort in older adults. However, little research has been done regarding the racial and ethnic differences in the prevalence of MGD, and few studies have reported the MGD prevalence rates in older age groups. We conducted a retrospective chart review to investigate the prevalence of MGD in older adults of various ethnic and racial populations in a large academic healthcare network.

METHODS: Healthcare records of living patients aged 50 and older that had a clinical encounter within the UNC Ophthalmology system were obtained from the Carolina Data Warehouse for Health. This study encompassed patient records from April 4, 2014 to July 23, 2017. ICD and EMR codes for MGD and MGD spectrum disorders were used to obtain demographic information from this data. Subjects were categorized by sex, age, ethnicity, and race. Prevalence rates and 95% confidence intervals for each race, ethnicity, and age interval were calculated.

RESULTS: Out of 19,314 eligible patients who were seen at UNC Ophthalmology clinics, 2,002 patients were diagnosed with MGD. The highest prevalence rates of MGD were reported in Asians (10.20%; 95% CI, 7.58-13.59) and Caucasians (13.20%; 95% CI, 12.60-13.83), whereas African Americans displayed the lowest prevalence rate (5.56%; 95% CI, 4.87-6.34). With increasing decades of age, prevalence rates significantly increased from 7.72% (95% CI, 7.06-8.43) in the 5059 age group to 13.71% (95% CI, 12.33-15.24) in the 80-89 age group.

CONCLUSIONS: MGD varies in prevalence based on race and ethnicity, while also increasing with age. The likelihood of developing MGD with increasing age will be a potential focus for future study.

Further investigation is needed to delineate the association between race and ethnicity and the risk of developing MGD.

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Finding an Optimal Corneal Xenograft: Comparative Analysis of Corneal Matrix Proteins Across Species Yelin Yang1,2, Roholah Sharifi1, Yashar Adibnia1, Miguel Gonzalez-Andrades1

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Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA; 2University of Toronto Department of Ophthalmlogy and Visual Sciences, Toronto, Canada 1

BACKGROUND: The shortage of donor corneas worldwide has led to an increased interest in xenotransplantation, or using corneas from animals for transplantation into humans. One of the major challenges to success of transplant is the immune response from antigenicity of corneal proteins, which is likely the source of donor host reaction. It is thus important to find the species with corneal stromal protein composition that is most similar to humans to maximize success of the transplant. We aim to investigate the differences in corneal matrix proteins between various animal species compared to human.

METHODS: Species that were most commonly used for studies in clinical applications of corneal transplant were included in the study: pig, rabbit, guinea pig, chick, cat, dog, rat, mice, bovine, sheep, goat, horse, and zebrafish. From literature review, we identified 18 most abundant proteins present in the corneal stroma including collagens, proteoglycans (decorin, lumican, keratocan, biglycan), MAM domain containing protein 2 (MAMDC2), prolargin and vimentin. The corresponding protein for each species was obtained through PUBMED protein database. The protein for each species was compared to human using BLAST (Basic Search Alignment Search Tool). A final weighted score of corneal stromal protein compared to human was calculated based on reported abundance of these proteins.

RESULTS: The pig was the most comparable to humans in 4 of 8 of the collagens examined. The cat and horse were most similar to humans in terms of proteoglycans, while chick and zebrafish were least similar. Most of the species had similar sequences of MAMDC2, prolargin and vimentin, with the exception of chick and zebrafish. In the final score, pig had the highest points (93% compared to humans) suggesting most similar composition in corneal stromal matrix proteins compared to human, while zebrafish, horse and chick had the lowest scores (72%, 84% and 85% respectively). The other species fell within a close range.

CONCLUSIONS: Pigâ&#x20AC;&#x2122;s corneal stroma matrix protein composition was the most similar compared to human overall and in terms of collagens. Selection of the right animal model may help to reduce antigenicity and improve outcomes of corneal xenografts.

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Topical Pigment Epithelium-Derived Factor Suppresses Corneal Epitheliopathy and Inflammation in Dry Eye disease Man Yu1, Jiaxu Hong1, Afsaneh Amouzegar1, Merle Fernandes1, Yihe Chen1, Takeshi Nakao1, Reza Dana1

Schepens Eye Research Institute of Mass. Eye and Ear, Harvard Ophthalmology, Boston, MA

1

PURPOSE: Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein widely expressed in different tissues, including the cornea. PEDF has been identified as an effective inhibitor of angiogenic and inflammatory responses. In this study, we aimed to investigate the effect of topical PEDF on disease severity and corneal inflammation in a mouse model of dry eye disease (DED).

METHODS: DED was induced in female C57BL/6 mice by placing them in the controlled environment chamber for 14 days. Mice were assigned to one of three groups (n=5 each), to which either 200ng/ml of recombinant PEDF (high dose), 20ng/ml PEDF (low dose) or PBS (control) was administered topically twice per day for 14 days. Corneal fluorescein staining (CFS) was performed to evaluate disease severity. Corneas and draining lymph nodes (dLNs) of different treatment groups were harvested at Day 14. Frequencies of CD45+ cells in the cornea, T helper 17 (Th17) and T regulatory (Treg) cells in dLNs, and level of Foxp3 expression by Tregs in dLNs were investigated using flow cytometry.

RESULTS: Both high and low doses of PEDF significantly decreased CFS scores of DED mice at day 7 compared with PBS-treated controls (high-dose vs. control P<0.001; low-dose vs. control P=0.002). At day 7, CFS score of mice treated with high-dose PEDF was significantly lower than those treated with low-dose PEDF (P=0.041). At day 14, high-dose PEDF was associated with a significant decrease in the frequencies of CD45+ cells in the cornea compared to PBS-treated controls (P=0.016). However, there was no significant difference in frequencies of Th17 and Treg cells and expression of Foxp3 by Tregs derived from dLNs among three groups.

CONCLUSIONS: Our data demonstrate that topical PEDF suppresses corneal inflammation and decreases the severity of DED.

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Protective Efficacy of Antibody to Poly-N-Acetyl Glucosamine (PNAG) Against Streptococcus pneumonia and Staphylococcus aureus Conjunctivitis

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Tanweer S. Zaidi1, Tauqeer H. Zaidi1, Zhao Ge1, Gerald B Pier1 Brigham and Women's Hospital, Harvard Ophthalmology, Boston, MA

1

PURPOSE: The ocular conjunctiva is highly susceptible to infectious agents, of which ~80% are viral. The remaining causes are bacterial, with Streptococcus pneumonia and Staphylococcus aureus being common causes. Little is known about the innate and adaptive immune effectors in this component of the eye and whether local or systemic antibody can gain access to ocular pathogens in this tissue and mediate effective immunity. We evaluated whether opsonic antibody to the conserved surface/capsular polysaccharide, poly-N-acetyl glucosamine (PNAG) was protective against bacterial conjunctivitis.

METHODS: To induce infection, S. aureus or S. pneumoniae was injected directly into the conjunctiva of 6-7 week old A/J mice. Four, 24 and 32 hours post-infection a human IgG1 monoclonal antibody (MAb) to PNAG or control IgG1 MAb were either injected into the conjunctiva or applied topically. Forty-eight hours post-infection eyes were scored for pathology daily, using a 0 to 4 scale, evaluating palpebral and bulbar conjunctivae for erythema, edema, and exudation.

After 48 h animals were euthanized and CFU/conjunctiva determined.

RESULTS: Both topical and injected antibody to PNAG markedly reduced bacterial levels in the conjunctivae and reduced conjunctival pathology. Histopathologic analysis of infected eyes showed severe edema and lymphocyte infiltration into eyes of mice treated with control MAb, while the conjunctiva of the anti-PNAG treated MAb showed only very mild lymphocyte infiltration.

CONCLUSIONS: Antibody to PNAG demonstrated therapeutic efficacy in models of S. pneumonia and S. aureus conjunctivitis, and this particular set of animal experiments further validated the capacity of such antibody to protect against divergent microbial species.

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Harvard Department of Ophthalmology

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Phosphorylation of Β-Catenin During In Vitro Human Corneal Endothelial Cell Expansion Wenlin Zhang1,*, Ricardo Frausto1, Alice Barrington1, Anthony Aldave1 Stein Eye Institute, Los Angeles, CA

1

*Email: w.zhang@jsei.ucla.edu PURPOSE: Wnt/β-catenin signaling is involved in embryonic development and the maintenance of adult tissue homeostasis. Wnt signaling has been implicated in corneal endothelial cell (CEnC) development and in vitro expansion by many researchers. The phosphorylation of β-catenin is the primary means of regulating its adhesion, self-degradation and transcription functions: for example, EGFR-PI3K-AKT pathway mediated ser552 phosphorylation of β-catenin promotes its transcriptional activity, whereas GSK3β mediated ser33/37/thr41 phosphorylation of β-catenin facilitates its degradation. Here we investigated the phosphorylation pattern of β-catenin during in vitro human CEnC primary expansion to identify the activated signaling pathways during in vitro expansion.

METHODS: Primary human CEnC (pHCEnC) cultures were established from corneas of 10 donors with a mean age of 22 years (range 15-30). CEnC were isolated using collagenase/M4M5 protocols as previously described. Passage 1 of pHCEnC were subject to immunofluorescence staining at 5%, 30%, 95% and 100% confluence. Subcellular localization of non-phosphorylated β-catenin (Ser33/37/Thr41) and phosphorylated β-catenin (Ser33/37/Thr41, Thr41/Ser45, Ser552 or Ser675) were assessed with co-immunostaining for N-cadherin (cell junctions) and DAPI. RESULTS: When pHCEnC were non-confluent (5% and 30%), non-phos-β-catenin showed punctate cytoplasm staining; when near to or at confluence (95% and 100%), non-phos-β-catenin co-localized with N-cadherin at the cell junctions. Ser33/37/Thr41 and Ser552 phos-β-catenin were located in the nucleus in both subconfluent and confluent cells, with the % of cells in which either was localized to the nucleus decreasing with increasing confluence (from 75% to 8% and 54% to 0.1%, respectively). When pHCEnC were near to or at confluence, the majority of Ser552 and Ser675 phos-β-catenin co-localized to the cell junctions with N-cadherin. Thr41/Ser45 phos-βcatenin was seen in the nucleus of only one or two cells near to or at confluence.

CONCLUSIONS: The co-localization of non-phos-β-catenin and Ser675 phos-β-catenin with N-cadherin in confluent pHCEnC indicates their active role in the cadherin-based adherens junction formation. The nuclear localization of Ser33/37/Thr41 phos-β-catenin and Ser552 phos-β-catenin in non-confluent pHCEnC that decreases significantly as the cells reach confluence suggest there is an initial activation, then inhibition, of Wnt transcriptional activity during in vitro pHCEnC primary expansion. The signaling pathways involved in mediating these processes need further investigation.

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Image: Fuchs dystrophy specimen showing fragmented mitochondria in the corneal endothelium. Taken by: Anne-Sophie Benischke, PhD