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Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2015-12-22 Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration Shariff, Sami Shariff, S. (2015). Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26398 http://hdl.handle.net/11023/2699 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration by Sami Shariff A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN CARDIOVASCULAR AND RESPIRATORY PHYSIOLOGY CALGARY, ALBERTA DECEMBER, 2015 © Sami Shariff 2015 Abstract The traditional paradigm of airway remodeling in asthma has held that remodeling occurs after many years of chronic inflammation. However, studies have confirmed that remodeling changes are observed in children even before the clinical diagnosis of asthma is established. There is now robust evidence to indicate that children with recurrent human rhinovirus (HRV)-induced wheezing episodes are at significantly increased risk of developing subsequent asthma. A feature of airway remodeling is increased airway smooth muscle (ASM) mass with a greater proXimity of the ASM to the subepithelial region, and we interrogated the hypothesis that HRV-induced alterations of airway epithelial cell biology might regulate ASM migration. We demonstrated that ASM chemotaXis is GPCR dependent, can be regulated by cAMP and is dependent upon CCL5 release by the epithelium post-HRV infection. These observations substantiate the growing body of evidence that links HRV infections to the subsequent development of asthma. ii Acknowledgements Undertaking this graduate project, moving back to Calgary, and all the changes it has brought would not have been possible if Richard had not allowed me to become his student after just one meeting with me. Your trust and belief in me has allowed me to overcome the challenges I faced throughout my degree and I cannot say thank you enough. To both Richard and David, you have been mentors and idols for me and have lit the pathway forward for several years. Thank you for all your support, guidance, and criticisms, because without them I would not be who I am today. It truly has been an honor to work with such outstanding scientists and I hope to be as successful in my future endeavors as you are currently in yours. To my committee members, thank you for your time and your input. To all the members of the Leigh, Proud, Newton, Giembycz and Kelly labs: thank you. Your encouragement, advice, guidance, and support have made these past few years fly past. Shahina, thank you for being like an older sister and both pushing me and supporting me to achieve greater heights. Suzanne and Cora, thank you for your expertise and knowledge that made overcoming every challenge easier. Sergei, Chris, and Jason, thank you for being the best of friends and colleagues, and for providing consistent boosts to my morale. Lastly, thank you to my parents, my siblings, and my wife Aleena for understanding the challenges I faced and providing me with overwhelming support. I could not have made it here without your love. I hope to keep making you proud. iii Table of Contents Abstract .................................................................................................................................................... ii Acknowledgements ............................................................................................................................ iii Table of Contents ................................................................................................................................. iv List of Tables ...................................................................................................................................... viii List of Figures and Illustrations ...................................................................................................... ix 1 CHAPTER ONE: INTRODUCTION .............................................................................................. 1 1.1 Executive Summary .......................................................................................................................... 2 1.2 Literature review .............................................................................................................................. 3 1.3 Characteristics and definition of asthma .................................................................................. 4 1.4 Airway Hyperresponsiveness ....................................................................................................... 5 1.5 Airway Inflammation in Asthma ................................................................................................. 8 1.6 Airway Remodeling .......................................................................................................................... 9 1.7 Origins of Airway Remodeling ................................................................................................... 11 1.8 Human Rhinovirus ......................................................................................................................... 13 1.9 The Airways ...................................................................................................................................... 17 1.10 Role of the epithelium in asthma .............................................................................................. 21 1.11 HRV Infection of the epithelium ................................................................................................ 22 1.12 The Airway Smooth Muscle ......................................................................................................... 30 1.13 Thesis Objectives ............................................................................................................................ 40 2 CHAPTER TWO: MATERIALS AND METHODS .................................................................. 41 2.1 Materials ............................................................................................................................................ 42 2.2 Isolation of HBE and ASM from Human Lung Tissue .......................................................... 44 iv 2.3 HRV-16 ............................................................................................................................................... 46 2.4 HRV-16 Infection of Bronchial Epithelial Cells ..................................................................... 48 2.5.1 GPCR Array ..................................................................................................................................................... 50 2.6 Fluorescence-Activated Cell Sorting (FACS) Analysis ......................................................... 51 2.7 Enzyme Linked Immunosorbent Assay (ELISA) ................................................................... 53 2.7.1 Measurement of CCL5 and CXCL10 protein release ..................................................................... 53 2.7.2 Measurement of CXCL8 protein release ............................................................................................. 54 2.8 Multiplex Chemokine Array Assay (64-Plex) ........................................................................ 56 2.9 Filtration of HBE cell supernatants .......................................................................................... 60 2.10 Migration of ASM ............................................................................................................................. 60 2.10.1 Migration in the micro chemotaXis chamber ................................................................................... 60 2.10.2 ChemotaXis via the XCELLigence Real Time Cell Analyzer (RTCA) system ........................ 62 2.10.3 Determination of chemotaXis versus chemokinesis ..................................................................... 68 2.10.4 ASM pertussis toXin treatment ............................................................................................................... 68 2.10.5 β-agonist treatment of ASM ..................................................................................................................... 69 2.10.6 ASM treatment with adenylyl cyclase agonist ................................................................................. 69 2.10.7 ASM treatment with 8-Br-adenosine 3′, 5′-cyclic monophosphate (cAMP) ....................... 70 2.10.8 CCL5 inhibition in conditioned medium ............................................................................................ 71 2.10.9 Quantification of cell viability ................................................................................................................. 72 2.11 Data management ..........................................................................................................................
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