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Clinical Study of Canine Tear Lacritin As a Treatment for Dry Eye Katherine E

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Clinical Study of Canine Tear Lacritin As a Treatment for Dry Eye Katherine E James Madison University JMU Scholarly Commons Senior Honors Projects, 2010-current Honors College Spring 2016 Clinical study of canine tear lacritin as a treatment for dry eye Katherine E. Kelly James Madison University Follow this and additional works at: https://commons.lib.jmu.edu/honors201019 Part of the Amino Acids, Peptides, and Proteins Commons, Biotechnology Commons, and the Small or Companion Animal Medicine Commons Recommended Citation Kelly, Katherine E., "Clinical study of canine tear lacritin as a treatment for dry eye" (2016). Senior Honors Projects, 2010-current. 169. https://commons.lib.jmu.edu/honors201019/169 This Thesis is brought to you for free and open access by the Honors College at JMU Scholarly Commons. It has been accepted for inclusion in Senior Honors Projects, 2010-current by an authorized administrator of JMU Scholarly Commons. For more information, please contact [email protected]. Clinical Study of Canine Tear Lacritin as a Treatment for Dry Eye _______________________ An Honors Program Project Presented to the Faculty of the Undergraduate College of Integrated Science and Engineering James Madison University _______________________ by Katherine Elizabeth Kelly May 2016 Accepted by the faculty of the Department of ISAT, James Madison University, in partial fulfillment of the requirements for the Honors Program. FACULTY COMMITTEE: HONORS PROGRAM APPROVAL: Project Advisor: Robert McKown, Ph. D. Bradley R. Newcomer, Ph.D., Professor, ISAT Director, Honors Program Reader: Ronald Raab, Ph. D. Professor, ISAT Reader: Stephanie Stockwell, Ph. D. Assistant Professor, ISAT PUBLIC PRESENTATION This work is accepted for presentation, in part or in full, at [venue] The ISAT Senior Capstone Symposium on [date] April 15th, 2016 . TABLE OF CONTENTS List of Figures ............................................................................................................................................ 2 List of Tables ............................................................................................................................................. 2 Acknowledgements ................................................................................................................................ 3 Abstract ....................................................................................................................................................... 4 Introduction .............................................................................................................................................. 5 Background ........................................................................................................................................... 5 Summary of Previous Work ............................................................................................................ 6 Methods and Materials ....................................................................................................................... 11 Tear Collection and Elution ......................................................................................................... 11 Total Tear Protein Concentration ............................................................................................. 11 Indirect ELISA Assay ....................................................................................................................... 11 Antibody Specificity and Titrations .......................................................................................... 12 SDS Polyacrylamide Gel Electrophoresis and Western Blots .......................................... 13 Results ...................................................................................................................................................... 15 Total Tear Protein Concentration ............................................................................................. 15 Antibody Specificity and Titrations .......................................................................................... 17 Indirect ELISA Assay ....................................................................................................................... 19 Western Blots .................................................................................................................................... 22 Discussion ............................................................................................................................................... 26 Bibliography ........................................................................................................................................... 28 Appendix A- BCA Raw Data ............................................................................................................... 30 Appendix B- ELISA Raw Data ............................................................................................................ 38 Appendix C- ISAT Senior Symposium Presentation ................................................................. 59 1 LIST OF FIGURES Figure 1. SDS PAGE and western blot anaysis of tears and samples.. ................................... 7 Figure 2. RT-PCR of canine lacritin gene, purification of recombinant canine lacritin, and DEAE chromatography elution profile. ................................................................................... 8 Figure 3. Summary of indirect ELISA analysis. ............................................................................. 9 Figure 4. Western Blot analysis of normal and dry eye tears. .............................................. 10 Figure 5. BCA Standard curve. ......................................................................................................... 15 Figure 6. Titration of 6924 antibodies. ......................................................................................... 18 Figure 7. Checkerboard titration of 6924 antibodies.. ........................................................... 19 Figure 8. Standard Curve using 6924 antibodies. .................................................................... 20 Figure 9. Western blot analysis of dry eye and normal samples. ........................................ 23 Figure 10. Dry eye and normal total tear protein as a function of STT values. .............. 24 Figure 11. Dry eye and normal lacritin concentrations as a function of STT values. .. 25 LIST OF TABLES Table 1. Summary of total tear protein in dry eye canines. .................................................. 16 Table 2. Summary of total tear protein in normal canines. .................................................. 17 Table 3. Summary of lacritin concentrations in dry eye canines. ....................................... 21 Table 4. Summary of lacritin concentrations in normal canines. ....................................... 22 2 ACKNOWLEDGEMENTS I would like to thank advisor Dr. Robert McKown for giving me the opportunity to conduct this research and for supporting me throughout this process. Dr. McKown has always been there to offer help and answer any questions I may have with patience and kindness. I would also like to thank my readers, Dr. Ron Raab and Dr. Stephanie Stockwell for their support, input and advice. I would like to give a special thanks to my collaborator Dr. Julie Disney for all the work she contributed and being such a pleasure to work with. I would like to thank all my lab mates, especially Eliza Gaylord, who was always willing to lend a helping hand in the lab. Lastly, I would like to extend a thank you to my parents for supporting me and my decision to come JMU and my friends who have always encouraged me to achieve my goals. 3 ABSTRACT Keratoconjunctivitis sicca (KCS), the deficiency of tears also known as dry eye, is a prevalent disease that affects both humans and canines. The current treatment for dye eye, cyclosporine (Restatis®), only provides temporary relief, is often associated with discomfort and is inconsistently effective. Lacritin is a naturally occurring tear glycoprotein secreted from the human and canine lacrimal glands. It has been shown that lacritin stimulates basal tearing in rabbits when applied topically. This study characterized the amount and form of lacritin found in the tears of dogs with healthy and dry eyes— information which may be applied to the development of a lacritin-inspired therapeutic for humans and canines. In collaboration with the Virginia-Maryland College of Veterinarian Medicine, tear samples were collected from dogs being treated at the veterinary clinic and transported to JMU for analysis. At JMU, the lacritin and other proteins found in the canine tears were analyzed by indirect Enzyme-Linked Immunosorbent Assay (ELISA), SDS- Polyacrylamide Gel Electrophoresis (SDS-PAGE), and western blot. A total of 64 tear samples were analyzed with 32 samples from healthy dogs and 32 samples from dogs clinically diagnosed with dry eye. ELISA revealed that canines with KCS had a significant decrease in tear lacritin. Western blot analysis detected prominent bands in healthy tears at approximately 18 kDa (corresponding to monomeric canine lacritin) that were absent or faintly observed in tears from dry eye dogs. This study provides clinical data reinforces the hypothesis that lacritin replacement may be an effective therapeutic for dry eye. 4 INTRODUCTION Background Keratoconjunctivitis sicca (KCS), the deficiency of tears also known as dry eye, is a prevalent disease that affects both humans and canines. Symptoms
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