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Determining the feasibility of assessing salivary biomarkers and swallowing perception in those with… Letawsky, Veronica Helen 2019

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DETERMINING THE FEASIBILITY OF ASSESSING SALIVARY BIOMARKERS AND SWALLOWING PERCEPTION IN THOSE WITH SJOGREN’S SYNDROME AND HEALTHY CONTROLS by  Veronica Helen Letawsky  B.Sc., The University of Alberta, 2011 Dip.Ling., The University of British Columbia, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Audiology and Speech Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2019  © Veronica Helen Letawsky, 2019   ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  Determining the Feasibility of Assessing Salivary Biomarkers and Swallowing Perception in those with Sjogren’s Syndrome and Healthy Controls  submitted by Veronica H. Letawsky in partial fulfillment of the requirements for the degree of Master of Science in Audiology and Speech Sciences  Examining Committee: Dr. Stacey Skoretz, School of Audiology and Speech Sciences, Faculty of Medicine Supervisor  Dr. Camilla Dawson, University Hospitals Birmingham NHS Foundation Trust Supervisory Committee Member  Dr. Caroline Nguyen, Department of Oral Health Sciences, Faculty of Dentistry Supervisory Committee Member  Additional Examiner   Additional Supervisory Committee Members:  Supervisory Committee Member  Supervisory Committee Member    iii Abstract  Sjogren’s syndrome (SS) is an autoimmune disease characterized by altered saliva quantity and composition. Saliva is integral to swallowing, contributing to bolus formation, lubrication, and digestion. Salivary changes in SS can affect the perceptions and execution of deglutition resulting in increased residue and impaired bolus transport and formation. To date, no study has investigated the link between salivary biomarkers and swallowing perceptions. Our objectives were: 1) to explore the operational feasibility of investigating saliva quantity (volume) and quality (proteins/hormones) in those with and without SS, and 2) to explore the relation between saliva and swallowing perceptions across participants. We conducted a matched case-control, mixed methods study, collecting feasibility data and conducting quantitative and qualitative measures. We collected unstimulated and stimulated whole saliva conducting sialometric (flow rate) and sialochemical analyses including α-amylase, cortisol, mucins (MUC5B, MUC7), C-reactive protein (CRP), and total protein. We measured oral dryness (Clinical Oral Dryness Score [CODS]) and swallowing perception (SWAL-QOL Survey). We described the data using means (±SD) and medians (IQR), compared between groups using t-tests and Mann-Whitney U, as appropriate. We explored Pearson correlations comparing salivary data with oral dryness and swallowing perception. Over 13-weeks, we enrolled a convenience sample of 12 (N): cases (n1) = 6, controls (n2) = 6 with five females, one male per group. Ages ranged from 31 to 68 years (n1, primary SS) and 31 to 64 years (n2). All participants completed assessments and produced analyzable saliva. Those with SS presented with reduced flow rate (p = .003) and increased total protein, cortisol, and CRP (p < .01). Oral dryness correlated negatively with unstimulated flow rates (r = -.63, p = .03) and positively with total protein (r = .77, p = .003) and α-amylase (r = .83, p = .001) concentrations. Swallowing perception, specifically salivary lubricative properties,   iv negatively correlated with total protein (r = -.81, p = .001) and α-amylase (r = -.83, p = .001) concentrations. The study design was feasible as proposed. We were the first to determine correlations between salivary properties and swallowing perceptions in those with and without SS. We propose suggestions for future investigations.     v Lay Summary  Saliva is important for a healthy mouth and for swallowing. It consists of different proteins that can help with swallowing. Some diseases, like Sjogren’s syndrome (SS), cause changes in saliva including a reduced amount and thicker saliva. This can affect eating and drinking, in turn, making it difficult to get enough nutrition and have a healthy and happy lifestyle. Our study looked at whether we could examine the differences in saliva between those with and without SS and whether certain proteins were linked to how people perceive their swallowing. We found those with SS had less saliva and more proteins. Those with SS also showed more stress and inflammation when compared to healthy individuals. For everyone, we found a link between dry mouth and what was in the saliva, and how their swallowing was perceived. Our findings may inform future testing and treatment of swallowing difficulties in various diseases.     vi Preface  This thesis is the original work of V. H. Letawsky written under the supervision of Dr. S. A. Skoretz. Reviews of the thesis were conducted by the supervisory committee members, Drs. C. Dawson and C. Nguyen, and members of the Weinberg Lab, Drs. J. Weinberg and P. J. Holman.  The study design was developed by V. H. Letawsky and S. A. Skoretz with contributions from C. Dawson, P. J. Holman, C. Nguyen, and J. Weinberg. The study was conducted at the Swallowing Innovations Lab and the Weinberg Lab. Recruitment, enrolment, consent, and study participation were completed by V. H. Letawsky at the Swallowing Innovations Lab. Salivary analyses were conducted at the Weinberg Lab by V. H. Letawsky, P. J. Holman, W. Yu, and Dr. T. Bodnar. Statistical analyses were conducted by V. H. Letawsky, P. J. Holman, and S. A. Skoretz.  This research was approved by the Clinical Research Ethics Board through the University of British Columbia on November 19, 2018. The certificate number assigned was H18-02328.      vii Table of Contents  Abstract ................................................................................................................................... iii Lay Summary ............................................................................................................................v Preface ..................................................................................................................................... vi Table of Contents ................................................................................................................... vii List of Tables .............................................................................................................................x List of Abbreviations ............................................................................................................... xi Acknowledgments ................................................................................................................. xiii Dedication ................................................................................................................................ xv Chapter 1 : Introduction .......................................................................................................1 1.1 Pathophysiology of Sjogren’s Syndrome ......................................................................3 1.2 Saliva’s Role in Swallowing ........................................................................................4 1.3 Salivary Changes in Sjogren’s Syndrome .....................................................................6 1.4 Swallowing in Sjogren’s Syndrome .............................................................................8 1.5 Literature Gaps .......................................................................................................... 10 1.6 Objectives .................................................................................................................. 11 1.7 Significance of Study ................................................................................................. 12 Chapter 2 : Methods............................................................................................................ 13 2.1 Study Process ............................................................................................................ 13 2.2 Participants ................................................................................................................ 14 2.3 Recruitment ............................................................................................................... 14 2.4 Consent and Enrolment .............................................................................................. 14 2.5 Feasibility Data Collection ......................................................................................... 15 2.6 Assessments .............................................................................................................. 16   viii 2.6.1 Quantitative Measures ........................................................................................ 16 Oral Speech Mechanism Screening Examination ....................................................... 16 Clinical Oral Dryness Score ....................................................................................... 16 Saliva Capture ........................................................................................................... 16 Sialometrics, Acidity, Aliquots, and Storage .............................................................. 17 Sialochemical Analyses ............................................................................................. 18 Demographics ............................................................................................................ 21 2.6.2 Qualitative Measure ........................................................................................... 21 SWAL-QOL Survey .................................................................................................. 21 2.7 Analyses .................................................................................................................... 21 2.7.1 Scoring and Descriptive Summaries ................................................................... 21 2.7.2 SWAL-QOL ...................................................................................................... 22 2.7.3 Exploratory Comparisons ................................................................................... 23 Chapter 3 : Results .............................................................................................................. 24 3.1 Study Feasibility ........................................................................................................ 24 3.1.1 Participant Recruitment and Characteristics ....................................................... 24 3.1.2 Operational Requirements .................................................................................. 27 3.2 Quantitative Measures ............................................................................................... 28 Oral Speech Mechanism Screening Examination ....................................................... 28 Clinical Oral Dryness Score ....................................................................................... 28 3.2.1 Salivary Analyses............................................................................................... 28 Sialometry & Acidity ................................................................................................. 28 Sialochemistry ........................................................................................................... 30 Total Protein .............................................................................................................. 30 Alpha-amylase, Cortisol, CRP, MUC5B, and MUC7 ................................................. 30 3.3 Qualitative Measure ................................................................................................... 33 SWAL-QOL .............................................................................................................. 33 3.4 Exploratory Comparisons .......................................................................................... 34 Comparing CODS and SWAL-QOL Scores to Salivary Data ..................................... 34 Comparing SWAL-QOL Domains and Symptoms to Salivary Data ........................... 34   ix Chapter 4 : Discussion......................................................................................................... 39 4.1 Study Feasibility ........................................................................................................ 39 4.2 Salivary Biomarkers: Sialometric and Sialochemical Analyses .................................. 42 4.3 Salivary Biomarkers and Perceptions of Swallowing ................................................. 46 4.3.1 Perceiving Oral Dryness ..................................................................................... 46 4.3.2 Quality of Life as it Relates to Swallowing ........................................................ 48 4.4 Limitations ................................................................................................................ 51 4.5 Clinical Implications and Future Directions ............................................................... 52 4.6 Conclusion................................................................................................................. 54 References................................................................................................................................ 55 Appendices .............................................................................................................................. 68 Appendix A: OSMSE-3 Scoring Form ............................................................................... 68 Appendix B: SWAL-QOL Survey ..................................................................................... 70 Appendix C: Recruitment Posters ...................................................................................... 75 Appendix D: Consent Forms ............................................................................................. 77 Appendix E: Table of Study Operational Requirements ..................................................... 89     x List of Tables  Table 2.1 Immunoassay Protocol According to Protein/Hormone .......................................... 19 Table 3.1 Participant Eligibility According to Recruitment Method ....................................... 25 Table 3.2 Recruitment Methods and Enrolment by Study Week ............................................ 26 Table 3.3 Summary of Participant Characteristics .................................................................. 27 Table 3.4 Sialometry and Acidity Results According to Collection Method and Group .......... 29 Table 3.5 Assay Results According to Collection Method ..................................................... 31 Table 3.6 SWAL-QOL Scores According to Domain and Participant Group.......................... 35 Table 3.7 SWAL-QOL Symptom Scores According to Participant Group ............................. 36 Table 3.8 Comparing CODS, SWAL-QOL, and Salivary Data .............................................. 38     xi List of Abbreviations α-amylase  Alpha-amylase µg   Microgram µL   Microlitre BCA    Bicinchoninic acid BSA   Bovine serum albumin CODS   Clinical Oral Dryness Score CRP   C-reactive protein DDK    Diadochokinesis dL   Decilitre EIA   Enzyme immunoassay ELISA  Enzyme-linked immunosorbent assay g   Gram or Unit of gravity h   Hours ID   Participant identifier IISBR   Institute for Interdisciplinary Salivary Bioscience Research IQR   Interquartile range L   Litre MANOVA  Multivariate analysis of variance min   Minutes mL   Millilitre MUC5B  Mucin 5B MUC7   Mucin 7 ng   Nanogram nm   Nanometer   xii NSB   Non-specific binding NSTEMI  Non-ST-elevation myocardial infarction OD   Optical density OSMSE-3  Oral Speech Mechanism Screening Examination-Third Edition pg   Picogram pH   Potential of hydrogen pSS   Primary Sjogren’s syndrome RA   Reducing agent RPM   Revolutions per minute SAS   Thesis supervisor SASS   School of Audiology and Speech Sciences SCA   Saliva collection aid SD   Standard deviation Si-Lab   Swallowing Innovations Lab SS   Sjogren’s syndrome sSS   Secondary Sjogren’s syndrome SWAL-QOL  Swallowing quality of life SWS   Stimulated whole saliva TMB   Tetramethylbenzidine U   Unit UBC   University of British Columbia UWS   Unstimulated whole saliva UWS-A  Unstimulated whole saliva via saliva collection aid UWS-B  Unstimulated whole saliva via swab VHL   Graduate student researcher W-Lab  Weinberg Lab    xiii Acknowledgments I am immensely grateful to my supervisory committee and so very proud to have had your select guidance. Thank you for so swiftly believing in me and cultivating the same belief in myself.    To my supervisor, Dr. Stacey Skoretz, you are the mentor of all mentors. I cannot express how privileged I feel that our paths in life and academia have crossed. It has been beyond meaningful to have your leadership within this research endeavour. Thank you for your patience with and welcoming of my endless questions, check-ins, worries, and the like, as well as for your pragmatic approach and sincere understanding of and interest in me not only as a student, but as an individual. I am so appreciative of your discernment; thank you for encouraging and pushing me when I needed it, while also knowing when I needed an ear or a shoulder. Your astute guidance and the certainty with which you have believed in my capacity has been profoundly impactful in completing my thesis. I extend my utmost gratitude to you for striving to embolden me in countless ways. I am honoured to have been your first thesis student and so very grateful to have had your guidance as we navigated a journey novel to us both. It has been an absolute privilege to learn from and along with you. I continue to be inspired by your intellect, dedication, and inimitable resilience in academia and beyond.   I am equally privileged to have been supported by Dr. Camilla Dawson (University Hospitals Birmingham NHS Foundation Trust) and Dr. Caroline Nguyen (UBC, Department of Oral Health Sciences). Thank you both for your willingness to serve on my committee and for the provision of expert guidance. Your confidence in my abilities and unwavering support has been incredibly meaningful. Thank you for your insights specific to my thesis and your unequivocal encouragement and reinforcement of future research endeavours.   I am graciously indebted to Dr. Joanne Weinberg and members of UBC’s Weinberg Lab: Dr. Parker Holman, Wayne Yu, and Dr. Tamara Bodnar, whose manuscript reviews, laboratory expertise, and thorough knowledge of salivary analyses were essential in completing this study.   I wish to extend my appreciation to the academic and clinical faculty (including recent emeriti), staff, and my fellow students at the School of Audiology and Speech Sciences (SASS).   Sincerest thanks to Dr. Valter Ciocca (SASS) for providing early review of the study protocol and Professor Vinidh Paleri (The Royal Marsden Hospital, London) for salivary insights.   The training I received at the University of California-Irvine’s Institute for Interdisciplinary Salivary Bioscience Research was invaluable to this study. I am expressly grateful to UBC’s Language Sciences Steering Committee who provided travel funding support.   Thank you to the associates of the Swallowing Innovations Lab who provided support, assistance, and company; including, but not limited to Julia Varanese who contributed to data review and Alyson Budd for valuable insights to participant recruitment.   Thank you to those that gave their time to participate in this study. I am especially grateful to those with Sjogren’s syndrome who were willing to share their unique experience.   xiv I am incredibly grateful for the friendship I have been so lucky to receive across provinces and throughout my education. In particular, I wish to acknowledge the circle of friends that I have grown in Vancouver and while studying at UBC. Your compassion, support, understanding, and especially your loyalty, have been remarkably meaningful.   I am thankful to my entire family; the closeness of ours is of such value and I am truly fortunate to have such extensive familial support. Many thanks to those who were dreaming big for me before I knew I was.   Thank you to my darling nephews, Berg and Case, for being the light when I couldn’t find it anywhere else. Your love and laughter is everything.   To my brothers, Peter and Billy, thank you for your understanding of my aspirations and for honouring and supporting my goals. Special thanks for motivating an interest in Sjogren’s syndrome and being open to this exploration.   I wish to express immeasurable gratitude to my parents, Mary and Wayne. Thank you for always emphasizing the importance of education and fostering my drive to succeed academically; to “kick some academic butt”, as you say. I am deeply appreciative of your support, in ways both moral and financial, throughout my prolonged academic journey. Your belief in my abilities and encouragement of my ambitions means the world. Thank you for having such faith in who I am.      xv Dedication  In memory of my Baba, Anna Sudyk, who so resolutely demonstrated love and pride for family, and whose own strength, courage, and grace remains a prodigious inspiration.  1 Chapter 1: Introduction Sjogren’s syndrome (SS), which occurs in approximately 1% of the population (Carsons, 2001), is an autoimmune disease affecting the exocrine system. The etiology of SS is considered multifactorial, varying across individuals (Garcia-Carrasco et al., 2006). Inflammatory triggers may include hormonal influences (Rogus-Pulia & Logemann, 2011), environmental factors (e.g., virus), and/or a genetic predisposition, specifically, a family history of autoimmune disorders (Amarasena & Bowman, 2007; Garcia-Carrasco et al., 2006). The prevalence of SS varies relative to sex and population (Reksten & Jonsson, 2014) with greater prevalence in females (9:1 female to male ratio), favouring those of Caucasian descent (Patel & Shahane, 2014). While it may occur at any age, it typically presents later in life between 40 and 60 years of age (Patel & Shahane, 2014; Reksten & Jonsson, 2014; Ruiz Allec et al., 2011). SS often goes unrecognized, resulting in diagnostic and treatment delay (Kassan & Moutsopoulos, 2004; Ruiz Allec et al., 2011). These challenges may be due to nonspecific systemic symptom presentation and overlap with other diseases and/or medication side effects (Patel & Shahane, 2014). While diagnosis remains challenging, diagnostic modalities have been refined (Kassan & Moutsopoulos, 2004) to facilitate improved and earlier SS identification.  The disease is categorized as primary (pSS) when occurring alone, and secondary (sSS), which is more common, when occurring in conjunction with other autoimmune diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus; Reksten & Jonsson, 2014; Ruiz Allec et al., 2011). Diagnosis of SS follows an international classification system (Vitali et al., 2002), though typically requires at least two of the following: (1) dry eyes, (2) lip biopsy confirming lymphocytes as the cause of dry mouth, or (3) associated extraglandular connective tissue disease (Rogus-Pulia & Logemann, 2011). Formal classification criteria include subjective (i. ocular   2 symptoms, ii. oral symptoms) and objective (iii. ocular signs, iv. salivary gland histopathology, v. diagnostic confirmation of salivary gland involvement, and vi. presence of autoantibodies) parameters (Vitali et al., 2002). Criterion item-based classification rules and exclusion criteria determine the identification of pSS and sSS. Exclusion criteria are: past head and neck radiation, pre-existing lymphoma, graft-versus-host disease, sarcoidosis, acquired immunodeficiency disease, hepatitis C, and the use of anticholinergic drugs (since time <4-fold drug half-life; Vitali et al., 2002). When differentiating between primary and secondary forms, pSS is defined by: (a) the presence of any four criteria with either criterion iv. or criterion vi. included and positive, or (b) the presence of any three objective criteria (iii., iv., v., and/or vi.; Vitali et al., 2002). In those with an associated disease, a diagnosis of sSS may follow from the presence of criterion i. or ii., along with any two of criteria iii., iv., or v. (Vitali et al., 2002).  A ‘sicca complex’ characterizes the disease with a reduction in both tears and saliva, resulting in dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia) with or without hyposalivation (i.e., reduction of both salivary volume and flow rate; Garcia-Carrasco et al., 2006; Rogus-Pulia & Logemann, 2011). The xerostomia is a result of salivary gland destruction and glandular dysfunction (Fox & Stern, 2002). In addition, those with SS present with extraglandular systemic features (e.g., fatigue, arthritis) and complications (e.g., dental caries, infections, risk of B-cell lymphoma; Garcia-Carrasco et al., 2006). Salivary gland hypofunction often manifests in the early stages; however, as the disease progresses, salivary gland dysfunction may stabilize, though other limitations may ensue as a result of the disease (Pijpe et al., 2007). These oral features result in difficulties with oral intake, speaking, voice, and swallowing (Atkinson, 1993; Pierce et al., 2016; Ruiz Allec et al., 2011).   3 Treatment for SS is aimed at symptom management (Amarasena & Bowman, 2007). For example, oral dryness can be managed with oral moisturizers and saliva substitutes (Aliko, Alushi, Tafag, & Isufi, 2012; Rhodus & Bereuter, 2000), frequent liquid intake, or chewing gum (Carr et al., 2012). In those with residual salivary gland function, cholinergic agonists (e.g., Pilocarpine) may also be used to stimulate muscarinic receptors for subsequent salivation (Amarasena & Bowman, 2007; Carr et al., 2012). While considered a benign disease, it is the secondary complications (e.g., lymphoma risk) and associated diseases of SS that have the potential to be life-threatening (Kassan & Moutsopoulos, 2004). 1.1 Pathophysiology of Sjogren’s Syndrome  This chronic, systemic inflammatory disorder occurs secondary to lymphocytic infiltration of the epithelia (Patel & Shahane, 2014) and exocrine glands, primarily the lacrimal and salivary glands (Fox & Stern, 2002; Garcia-Carrasco et al., 2006; Rogus-Pulia & Logemann, 2011). Dendritic, B-, and T-cell infiltration destroys the lacrimal and salivary glands, increases cytokine secretion, interferes with muscarinic reception, and obstructs cell interactions leading to oral mucosa inflammation (Garcia-Carrasco et al., 2006). Autoantibody and cytokine involvement, characteristic of autoimmune disease, also occurs (Reksten & Jonsson, 2014). Specifically, antibodies to the anti-Ro/SSA and anti-La/SSB proteins contribute to SS diagnostic criteria (Garcia-Carrasco et al., 2006), whereas elevated cytokine levels (Reksten & Jonsson, 2014) interrupt secretory signaling in the salivary glands (Proctor, 2016). Autoantibody interaction with muscarinic receptors affects parasympathetic neurotransmission to the salivary gland acinar cells (Berra, Sterin-Borda, Bacman, & Borda, 2002). These cells have a role in the initiation of saliva secretion (Cankaya et al., 2010). Collectively, these factors contribute to the   4 systemic changes in salivation, namely, reduced salivary volume/flow rate and altered composition, which thereby impact deglutition. 1.2 Saliva’s Role in Swallowing In general, saliva supports the maintenance of oral health and deglutition (Matsuo & Palmer, 2013; Tiwari, 2011). Specifically, saliva possesses enzymatic and antimicrobial properties aiding in digestion and preventing dental caries and decay, respectively (Carpenter, 2013; Dawes, et al., 2015; Humphrey & Williamson, 2001; Matsuo & Palmer, 2013; Tiwari, 2011). In addition, saliva’s buffering attributes and mild acidity (pH of 6.0 to 7.0) is central to dental caries prevention (Humphrey & Williamson, 2001; Kaplan & Baum, 1993). During deglutition, saliva contributes to bolus formation and cohesion during the oral swallowing phases (Dawes et al., 2015; Pedersen, Bardow, Beier Jensen, & Nauntofte, 2002). Initiation of the swallow occurs shortly after peak bolus cohesion is attained (Pedersen et al., 2002; Prinz & Lucas, 1995; 1997). Additionally, saliva’s lubricative properties reduce friction during bolus formation and transfer (Bongaerts, Rosetti, & Stokes, 2007; Dawes et al., 2015). This ensures minimal bolus residual particulates remain following the swallow and effective bolus transfer throughout the upper digestive tract.  Saliva is produced in a variety of locations in the upper airway with its composition varying according to origin. The major paired salivary glands are the sublingual, submandibular, and parotid (Pedersen et al., 2002). Minor salivary glands are also located throughout the submucosa of the oral cavity and tongue (Carpenter, 2013). For healthy individuals, daily production of whole saliva ranges from 0.5 to 1.5 L (Pedersen et al., 2002). At rest, the submandibular glands contribute the most to whole saliva (~65%) with other contributions as follows: parotid (~20%), minor (<10%), and sublingual (~7%; Humphrey & Williamson, 2001).   5 Secretion types vary across the glands: strictly serous fluids are secreted by the parotid glands with mucous secreted by most minor glands (serous via minor von Ebner’s glands; Carpenter, 2013; Dawes et al., 2015). Mixed secretions, comprised of both mucous and serous fluids, are secreted by the sublingual and submandibular glands (Humphrey & Williamson, 2001).  While food visualization may trigger salivation, primary stimulation of oral secretions is due to gustation and mastication (Carpenter, 2013). This stimulated saliva, comprises the majority (80%+) of all oral secretion (Humphrey & Williamson, 2001), with significant contribution from the parotid glands (Carpenter, 2013). Variations in flow rate have a direct impact on swallowing. The rate by which saliva flows varies according to condition (stimulation) and environment (e.g., hydration, medication effects). The minimum accepted flow rates for unstimulated (resting) and stimulated whole saliva are 0.1 mL/min and 0.2 mL/min (Humphrey & Williamson, 2001), respectively, with unstimulated rates below 0.1 mL/min (Dawes, 2008) and stimulated rates below 0.7 mL/min (Hopcraft & Tan, 2010) considered hyposalivation. Average unstimulated flow rates range between 0.3 and 0.4 mL/min (Dawes, 2008). Saliva varies not only by origin and flow rate but also in its protein/hormone composition. Proteins contribute to salivary lubricative and viscoelastic properties (Zussman, Yarin, & Nagler, 2007) while aiding with deglutition. In addition, protein concentrations correlate with perception, relative to food texture (Engelen et al., 2007). These proteins include mucins which are found in saliva as well as on a mucosal oral film (Carpenter, 2013). They provide lubrication and cohesive properties during swallowing (Carpenter, 2013), specifically during mastication and bolus formation (Dawes et al., 2015), as well as sensory perception (Vijay, Inui, Dodds, Proctor, & Carpenter, 2015), with mucin levels positively correlated with rheological properties (Chaudhury, Shirlaw, Pramanik, Carpenter, & Proctor, 2015; Inoue et al., 2008; Park, Chung,   6 Kim, Chung, & Kho, 2007). There are three main mucins expressed in saliva; transmembrane mucins (e.g., MUC1) and two soluble secreted mucins (Castro et al., 2013): high molecular weight MUC5B (gel forming) and low molecular weight MUC7 (Almstahl, Wikstrom, & Croenik, 2001; Chaudhury et al., 2015). MUC5B, the main salivary mucin (Castro et al., 2013), is a potentially more effective lubricant (Aguirre et al., 1989); however, a major role in oral lubrication has been postulated for both MUC5B and MUC7. These lubricative qualities are due to mucins’ low solubility, high elasticity, viscosity, and adhesiveness (Castro et al., 2013).  Parotid gland saliva contains other essential proteins, including amylase, its most prominent salivary protein (Carpenter, 2013; Humphrey & Williamson, 2001). With this abundance of amylase, its enzymatic form, alpha-amylase (α-amylase), dominates in the saliva of those with typical health and has numerous attributes including that of a marker of sympathetic nervous system activity (Granger, Kivlighan, El-Sheikh, Gordis, & Stroud, 2007) and “fight or flight” responsivity (Gordis, Granger, Susman, & Trickett, 2008). Unlike other salivary components, α-amylase is directly produced by the salivary glands (Granger et al., 2007), providing a measure specific to the oral cavity. However, flow rate, diurnal rhythms, and the stress response can alter salivary α-amylase levels (Beltzer et al., 2010; Granger et al., 2007). With respect to oral intake, this enzyme is crucial to the early digestion and breakdown of carbohydrates and starch (Carpenter, 2013; Dawes et al., 2015; Granger et al., 2007). As with mucins, α-amylase also has lubricative properties (Aguirre et al., 1989), a role in bolus clearance following mastication (Carpenter, 2013), and contributions to bolus perception.  1.3 Salivary Changes in Sjogren’s Syndrome Many salivary attributes are impacted by immune functioning. Those with SS experience reductions in saliva quantity (Rhodus, Colby, Moller, & Bereuter 1995a; Rhodus, Moller, Colby,   7 & Bereuter, 1995b), as measured by saliva flow rates (sialometrics), and saliva composition changes, specifically, sialochemical properties (Engelen et al., 2007; Kalk et al., 2001; 2002; Mathews, Kurien, & Scofield, 2008). While hyposalivation and flow rate reduction may contribute substantively to the perception of xerostomia, there may be other influential factors. Salivary flow rate can be inversely correlated with protein concentration, with greater flow rates leading to protein dilution (Engelen et al., 2007). As such, the reduced salivary flow rate in SS corresponds with a higher salivary protein concentration contributing to variable opacity, viscosity, and thickness (Fox, 2007). In addition, at emergent stages of the disease, salivary gland dysfunction leads to early changes in saliva quality (Mathews et al., 2008). These changes, which tend to arise prior to salivary gland hypofunction and detectable reductions in flow rate, are indicative of the loss of secretory cells following autoimmune attack and inflammation (Kalk et al., 2001). Therefore, altered saliva quality need be studied along with reduced quantity in SS, particularly in relation to deglutition.   The composition of saliva in those with SS differs from that of healthy controls in terms of proteins and enzymes, indicating a unique salivary biomarker signature (Tzioufas & Kapsogeorgou, 2015). Specifically, when compared to healthy controls, those with SS have fewer acinar proteins and higher inflammatory protein levels (Ferraccioli et al., 2010). Notably, salivary gland acinar cells produce 85-90% of salivary proteins (Kaplan & Baum, 1993), yet it is the acinar cells, along with ductal cells, that tend to incur damage in SS (Mahoney & Spiegel, 2003). In addition, C-reactive protein (CRP), a general marker of inflammation (Jonsson, Skarstein, Jonsson, & Brun, 2007), has been detected at varied levels in those with SS (Delaleu et al., 2015). Other salivary components, particularly α-amylase, are detected in lower levels in those with pSS as compared to sSS and controls (Fleissig et al., 2009). Though salivary cortisol   8 has been found not to differ between those with and without SS (Miller et al., 2012), such findings need be interpreted in relation to diurnal rhythms. In addition, given the intrinsic relation between α-amylase and cortisol, and the responsiveness of cortisol to acute environmental stressors, both warrant consideration in SS salivary profiling (Adam, Hoyt, & Granger, 2011; Gordis, Granger, Susman, & Trickett, 2006; Gordis, et al., 2008). Furthermore, these changes to salivary composition have implications for perception within oral intake and deglutition. This varied salivary profile and its perceptual counterparts have the potential to assist in the diagnosis and treatment of dysphagia (swallowing dysfunction) in those with SS. As such, investigating the interplay between saliva quantity and quality and its impact on swallowing function and perception is essential to further our understanding of patient experiences during this disease progression. 1.4 Swallowing in Sjogren’s Syndrome Oropharyngeal and esophageal dysphagia have been reported in those with SS (Amos, Baron, & Rubin, 2016; Pierce et al., 2016). While the primary etiology of dysphagia in those with SS is multifactorial, predominant factors are salivary volume and flow. While subjective reports of swallowing dysfunction have been correlated with lower salivary flow rates (Hughes et al., 1987), explicit correlations between low flow rate and swallowing physiology are broad-based. Physiological findings linked to saliva reduction include: post-swallow pharyngeal wall and vallecula residue (Eyigor et al., 2017), difficulties swallowing dry foods (Pijpe et al., 2007), and increased water consumption (Gonzalez, Sung, Sepulveda, Gonzalez, & Molina, 2014). These symptoms may be treated with oral moisturizers or saliva substitutes. Improvement in relation to hyposalivation, xerostomia, and swallowing, specifically mastication, have been noted   9 with their use (Aliko et al., 2012; Alves, Motta, Messina, & Migliari, 2004; Rhodus & Bereuter, 2000).  As with observable clinical signs and perceived symptoms, physiological findings of dysphagia also vary. Generally, those with SS exhibit more oral residue (Rogus-Pulia & Logemann, 2011), impaired bolus formation (as a result of salivary gland dysfunction; Kaplan, Zuk-Paz, & Wolff, 2008), and longer oral transit durations (Hughes et al., 1987). Additionally, pharyngeal phase impairments include pharyngeal residue, particularly in the vallecula (Rogus-Pulia & Logemann, 2011), most frequently with solid (Anselmino et al., 1997; Ruiz Allec et al., 2011) and semi-solid boluses (Eyigor et al., 2017). Similar to the oral phase, longer pharyngeal transit times have been documented (Anis & Soliman, 2013), characterized by reduced base of tongue retraction, prolonged hyoid movement and cricopharyngeal opening durations (Rogus-Pulia & Logemann, 2011). Yet to be determined systematically is the degree to which swallowing biomechanics are affected by salivary gland dysfunction (Kaplan et al., 2008). Both saliva quantity and quality play a key role in swallowing theoretically; however, the definitive connections between perception, saliva composition, and swallowing are only emerging. Some suggest salivary quantity fluctuations affect its lubricative properties and contingent perceptions, though this has not been widely investigated in those with SS (Rogus-Pulia, Gangnon, Kind, Connor, & Asthana, 2018). For example, reduced saliva production leads to inadequate lubrication, a perception of dry mouth, and a greater sense of effortful swallowing in relation to aging in healthy adults (Rogus-Pulia et al., 2018). Alternatively, in simulation studies, reduction in saliva production has a greater effect on residue than bolus lubrication and transport (Ho et al., 2017). As such, further inquiry into the association between swallowing perceptions and quantitative and qualitative aspects of saliva is necessary in the area of SS and beyond.   10 The ability to eat and drink (and their related social aspects) is inherently linked to quality of life metrics (Pierce et al., 2016). In SS, swallowing dysfunction has been associated with isolation (Reksten & Jonsson, 2014), depression, and a general reduction in quality of life (Eyigor et al., 2017). Additionally, lower levels of life quality and more severely impaired swallowing are associated with more severe forms of SS (Pierce et al, 2016). Specific to swallowing, those with SS have reported reduced life quality (Bolstad & Skarstein, 2016; Eyigor et al., 2017; Pierce et al., 2016; Reksten & Jonsson, 2014) secondary to hyposalivation and impaired oral sensation (Anis & Soliman, 2013; Rogus-Pulia & Logemann, 2011). While no correlation has been found between saliva quantity and perceived swallowing ability in those with and without SS (Rogus-Pulia & Logemann, 2011), altered salivary composition may play a role in the perceptions of impaired swallowing. Given that altered salivary protein profiles in those with SS exist, these altered profiles may impact swallowing sensation and perception, specifically as it relates to mechanisms most influenced by oral dryness (Field et al., 1997; Kaplan et al., 2008). Determining the cellular and physiological basis of perceived swallowing dysfunction in SS provides the opportunity to engage in proactive patient-specific treatment that targets symptoms early and maximizes quality of life. 1.5 Literature Gaps  While both quantitative and qualitative differences in the saliva of those with and without SS have been identified, no study has explored those measures in relation to perceptions of swallowing and life quality. Although saliva quantity has been investigated at length in this population, the reported impacts of saliva quantity on swallowing function remain largely nonspecific focusing on general volumetric analyses. Thus, exploration of perceptions of swallowing function and dry mouth is warranted in an effort to evaluate the relation between   11 altered saliva, perception, and quality of life. Though perceptions of swallowing function continue to be explored in relation to salivary changes in SS, to the best of our knowledge, no study has investigated the association of saliva quality (composition) and perceptions of swallowing in those with SS. 1.6 Objectives Our primary objective was to explore the operational feasibility of determining the amount and composition (i.e., protein/hormone concentrations) of saliva produced in individuals diagnosed with SS as compared to healthy controls. The secondary objective was to explore the relation between salivary composition and participants’ perceptions of swallowing. Our aim was to determine the feasibility of addressing the following research questions, when comparing those with SS to healthy individuals: (1) Do salivary production and protein/hormone concentrations as well as perception of swallowing differ? and (2) What is the relation between salivary production and protein/hormone concentrations and the perception of swallowing?   Quantitative (Rhodus et al., 1995a; 1995b) and qualitative (Engelen et al., 2007; Kalk et al., 2001; 2002; Mathews et al., 2008) differences in the saliva of those with SS have previously been identified. More specifically, changes in salivary volume and composition affect swallowing performance in SS (Eyigor et al., 2017; Gonzalez et al., 2014; Hughes et al., 1987; Pijpe et al., 2007; Zussman et al., 2007), and such differences may be observed upon comparison of cases to controls. In addition, altered perceptions of swallowing have also been recognized in those with SS (Rogus-Pulia et al., 2011; Rogus-Pulia & Logemann, 2018; Vijay et al., 2015). Accordingly, it was hypothesized that perceptions of swallowing would vary between cases and controls, which may correspond to differences between their salivary composition and volume (quality and quantity, respectively).   12 1.7 Significance of Study The following elements remain to be determined with respect to a comprehensive understanding of swallowing in SS: (1) explicit identification of syndrome-specific compositional changes in salivary components relevant to swallowing, (2) the precise impacts of altered saliva (quantity and quality) on the perception of swallowing in SS, and (3) the long-term implications of salivary changes and dysphagia on quality of life. In addition, prospective findings may elucidate SS-specific salivary biomarkers, as they apply to swallowing disorders. These biomarkers may prove instrumental in the differential diagnosis of dysphagia, affording early as well as individualized, patient-centered treatment. Ultimately, should we determine a link between salivary biomarkers and swallowing disorders, this approach may prove applicable to other patient populations including those with other autoimmune diseases, chronic illnesses, and head and neck cancers.     13 Chapter 2: Methods 2.1 Study Process  Using a case-control, mixed methods design, we conducted a feasibility study using quantitative and qualitative measures. In order to observe the structure and function of the oral cavity and determine xerostomia severity, we conducted a standardized examination of the oral cavity (Oral Speech Mechanism Screening Examination-Third Edition [OSMSE-3; St. Louis & Ruscello, 2000, Appendix A]) and an assessment of oral dryness severity (Clinical Oral Dryness Score [CODS; Osailan, Pramanik, Shirlaw, Proctor, & Challacombe, 2012]), respectively. We collected three saliva samples, including two samples of unstimulated whole saliva, each using a different collection device (saliva collection aid or SalivaBio oral swab; Salimetrics LLC, State College, PA), and one stimulated whole saliva sample through mastication of an oral swab. Sialometric analyses included salivary weight and flow rate, and sialochemical analyses included immunoassays to determine concentrations of the following analytes: α-amylase, cortisol, CRP, MUC5B, MUC7, as well as total protein content. To measure patient perception, we utilized a standardized, published questionnaire, the SWAL-QOL Survey (McHorney et al., 2002, Appendix B). We also collected demographic and medical history information from participants. Following University of British Columbia (UBC) Clinical Research Ethics Board approval (H18-02328), this study was conducted at the Swallowing Innovations Lab ([Si-Lab], UBC School of Audiology and Speech Sciences [SASS]) with salivary analyses conducted at the Weinberg Lab (W-Lab, UBC Department of Cellular and Physiological Sciences) with the assistance of Dr. Joanne Weinberg, Dr. Parker Holman, Wayne Yu, and Dr. Tamara Bodnar.   14 2.2 Participants   Through purposive sampling, case participants included individuals diagnosed with either pSS or sSS, 18 years of age or older, and fluent in English (written and oral). In view of other sources of salivary changes (outside of SS), we excluded those with a recent smoking history (within 10 years), prior history of radiotherapy or surgery for head and neck cancer, neurological and/or neurodegenerative disease, and current use of medications which result in hyposalivation. Following case enrolment, we matched healthy, non-smoking controls to cases according to age (± 5 years) and sex. 2.3 Recruitment We recruited all participants in Vancouver, BC, through two different means: site advertising and online/word of mouth. Site advertising included poster placement at UBC’s Friedman Building, campus health clinics (UBC Student Health Clinic, UBC Family Medical Clinic, University Village Clinic, Careville Medical Clinic), and St. Paul’s Hospital (Providence Health, Appendix C). Online methods included posting recruitment documentation to the SASS webpage and social media (Facebook, Twitter), the Si-Lab website, and Reddit pages (Vancouver, BC; Sjogren’s Syndrome). A UBC-hosted e-mail address was used for all contact. We initiated recruitment with cases, followed by matching with control participants.  2.4 Consent and Enrolment  Once eligibility was confirmed and we determined that participants met the study inclusion criteria, participants were consented and enrolled. A circadian rhythm has been observed in salivary flow rate with a peak in this rate occurring in the late afternoon (Dawes, 1972; Dawes et al., 2015) and the lowest flow during sleep (Dawes, 2008; Turner & Ship, 2007), which may carry over into the morning hours. As such, in effort to target optimal salivation, we   15 collected saliva samples from all participants after 1500h, at the participants’ convenience. So as to limit confounding effects, we scheduled and conducted the appointment of matched controls at the same time as their case counterpart (± 30 minutes). Prior to their appointment at the Si-Lab, we provided participants with an electronic consent form (Appendix D) and the SWAL-QOL questionnaire for their review.  Upon enrolment, we requested that participants utilizing oral moisturizers, saliva substitutes, and/or salivary replacement medications consult with their physician and, if permitted, temporarily discontinue the use of these products 48 hours prior to saliva collection (Rogus-Pulia & Logemann, 2011). Once at the appointment, we reviewed the consent form with participants, including details regarding participation, why the research was being conducted, what would happen during the study, and the possible benefits, risks, and discomforts. At that time, participants had an additional 30 minutes to decide whether or not to participate. Prior to consent, we asked general orientation questions related to three domains (name, place, and date) to confirm capacity. Once consented and enrolled, we linked participants’ information with a unique study identifier. All participation was voluntary, and participants had the option to withdraw from the study at any time without consequence. 2.5 Feasibility Data Collection Given the novel study design and emphasis on feasibility, which involved operationalizing population-specific saliva collection, feasibility parameters included: study recruitment (sources, enrolment, and rate), operational requirements (personnel, equipment, and laboratory time, and associated costs), and saliva collection (methods, instructions, and procedures).   16 2.6 Assessments 2.6.1 Quantitative Measures Oral Speech Mechanism Screening Examination  We conducted the OSMSE-3 with all participants according to the published protocol (St. Louis & Ruscello, 2000). We evaluated the appearance and non-speech functions of the lips, tongue, jaw, teeth, hard and soft palates, pharynx, and velopharyngeal mechanism, along with breathing and diadochokinesis (DDK; St. Louis & Ruscello, 2000). The DDK rates determine the speed and regularity of the movement of the oral articulators, along with motor planning (Duffy, 2013). Cumulative scoring across OSMSE-3 domains result in either a pass or fail outcome. Clinical Oral Dryness Score  We utilized the 10-point CODS scale (Osailan et al., 2012) to assess the severity of oral dryness. We evaluated the oral cavity of each participant on ten features of oral dryness including the appearance and adhesiveness of oral structures and saliva (Osailan et al., 2012). The scores from each feature are summed with higher totals indicating greater dryness severity. Saliva Capture Unstimulated and stimulated whole saliva samples were collected from all participants at the Si-Lab. To eliminate the influence of additional salivary stimulants and sources of altered protein/hormone concentration, we instructed participants to refrain from eating, smoking, chewing gum, brushing teeth, using dental floss, and drinking anything but water for 1.5 hours prior to saliva collection (Nguyen, Lin, Clark, Hovan, & Wu, 2018). The consumption of tap water was permitted up to 15 minutes prior to saliva capture, and we required participants to sit upright and refrain from speaking and swallowing during collection (Nguyen et al., 2018).    17 We collected resting or unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) from all participants for a minimum of two minutes up to a maximum of five minutes; however, participants were also permitted to determine stoppage timing based on their comfort. Participants were given a two-minute break in between each collection and sampling duration was recorded after each collection. First, unstimulated saliva was collected passively using two different collection devices: a straw-like saliva collection aid (Salimetrics, LLC, State College, PA) which fits into a 2 mL cryovial, and a SalivaBio oral swab (Salimetrics, LLC, State College, PA). Participants expectorated oral fluid every 30 seconds through the saliva collection aid, letting it drain into the attached cryovial (Navazesh & Christensen, 1982; Nguyen et al., 2018; Salimetrics, LLC, 2018) to provide the first unstimulated sample (UWS-A). For the second unstimulated sample (UWS-B), saliva was collected by placing a swab under the tongue and once saturated, the swab was placed into a swab storage tube (Salimetrics, LLC, 2018). Similarly, SWS was collected using a new swab, which participants placed into their mouth and were instructed to masticate. Once saturated, participants placed the swab into a new storage tube (Salimetrics, LLC, 2018). All samples were immediately lidded, labelled (with unique identifiers) and put onto ice for cooler transport to the W-Lab.  Sialometrics, Acidity, Aliquots, and Storage  Following transport to the W-Lab, we immediately weighed all samples using a laboratory balance, documented saliva weight in grams, and converted this value to millilitres (per equivalency of 1 g to 1 mL) to indicate volumetric quantity. We calculated salivary flow rate (millilitres per minute) based on the duration (minutes) for saliva collection. We then measured salivary acidity in UWS-A samples using pH indicator strips (pH range of 4.5 to 9.0; HealthyWiser®, China). Immediately following the sialometric analyses, we centrifuged the   18 saliva samples at 2190 g for 10 minutes at 4ºC and then prepared aliquots in 0.6 mL Eppendorf tubes labelled with unique identifiers. Minimum aliquot volumes were dependent on total saliva sample volume and determined in a manner to prevent more than one freeze/thaw cycle per analyte. Once aliquoted, we immediately lidded all tubes and stored them in a freezer at -25ºC.  Sialochemical Analyses Sialochemical analyses were performed in the W-Lab using enzyme-linked immunosorbent assay (ELISA) kits for mucins (MUC5B, MUC7; Cloud-Clone Corp., Katy, TX), α-amylase, cortisol, CRP (Salimetrics, LLC, State College, PA), and total protein content (bicinchoninic acid [BCA] protein assay; Thermo Fisher Scientific, Rockford, IL). Assay methods were conducted according to kit manufacturer standard protocols (Table 2.1), and testing for all saliva samples was completed simultaneously (per testing protein/hormone) and in duplicate, when saliva volume permitted. UWS-A samples were tested for α-amylase, cortisol, CRP, MUC5B, MUC7, and total protein content. The UWS-B and SWS samples were tested for α-amylase, cortisol, CRP, and total protein content. We documented salivary concentration for all proteins and hormones analysed. For assays, aliquoted samples were fully thawed, vortexed, and centrifuged for 15 minutes at 2190 g with any remaining sample volume immediately refrozen (Salimetrics, LLC, 2016; 2017). CRP assays were not run in duplicate due to limited sample volumes and prioritization of other proteins/hormones based on our research questions. As such, CRP assays were run in singlicate. For total protein, we analysed all samples at a two-fold dilution. For total protein outputs above the upper limit of detection, samples were re-run at a five-fold dilution (21-fold dilution for one sample with limited volume remaining).      19 Table 2.1 Immunoassay Protocol According to Protein/Hormone  Assay Kit Standard Protocol  α-amylase Cortisol CRP Total Protein MUC5B & MUC7      1) Reagents and microtiter plate brought to room temperature, plate layout and number of microtiter strips determined, and plate prepared with assay controls and saliva samples. 2) Plate reader set to incubate at 37˚C and α-amylase substrate heated (in covered trough). 3) Saliva samples diluted with α-amylase diluent to prepare a 1:10 dilution, mix, dilute further to final 1:200 dilution. 4) 8 µL of assay controls and saliva samples reverse pipetted into wells. 5) 320 µL preheated substrate added simultaneously to each well. 1) Reagents and microtiter plate brought to room temperature, plate layout and number of microtiter strips determined, two NSB wells selected, and 1x wash buffer prepared with 10x dilution (100 mL wash buffer concentrate, 900 mL deionized water). 2) Plate prepared with 25 µL of standards, controls, and saliva samples pipetted into individual wells (per layout). 25 µL of assay diluent pipetted into two wells (as zero) and two NSB wells. 3) 24 mL of assay diluent pipetted into disposable tube. 15 µL of enzyme conjugate added to assay diluent (1:1600 dilution). 4) Dilute conjugate solution mixed and 200 µL simultaneously added to each well. Plate mixed on rotator at 500 RPM for 5 minutes and incubated at room temperature (1 h).  1) Reagents and microtiter plate brought to room temperature, plate layout and number of microtiter strips determined, and 1x wash buffer prepared with 10x dilution (100 mL wash buffer concentrate, 900 mL deionized water). 2) CRP control and CRP standard reconstituted: 0.5 mL and 1 mL of deionized water, respectively. Room temp. incubation (20 min). 3) CRP dilution prepared: 150 µL CRP sample diluent and 150 µL 3000 pg/mL for 2x serial dilution of standard. 4) Saliva samples diluted 10x in CRP sample diluent (15 µL saliva, 135 µL diluent). 5) Per layout, 50 µL controls, standards, and diluted saliva samples added to wells. Two zero standard wells: 50 µL of CRP sample diluent. 6) 20 µL assay diluent added to disposable tube. 80 µL CRP antibody enzyme conjugate added to assay diluent for 1:250 dilution. 1) Standard control prepared: 200 µL of buffer and no RA 2) Sample control prepared: 200 µL of buffer and RA at sample concentration. 3) Protein standards prepared using buffer with no RA to dilute one BSA ampule. 4) Reconstitution buffer diluted 1:1 with water. 5) Saliva samples diluted 2x (re-run dilution factor: 5x; 21x) in lysis buffer.  6) 100 µL working reconstitution buffer added to compatibility reagent tube, stirred to dissolve, covered, and stored at 4˚C. 7) 9 µL standard and sample controls, standard, and samples added to each well. 8) 4 µL compatibility reagent solution added to each well. 1) Reagents and microtiter plate brought to room temperature, plate layout (7 standard wells, 1 blank) and number of microtiter strips determined 2) Stock solution diluted to 20 ng/mL. Standard reconstituted with 1 mL standard diluent (10 min., room temperature). 3) Seven tubes prepared with 0.5 mL standard diluent. Double dilution series prepared: diluted standard and 500 µL transfers. 4) Stock detection reagents A and B centrifuged, diluted to 100x working concentration. 5) 600 mL wash solution (1x) prepared with 20 mL wash solution concentrate diluted 30x with 580 mL deionized water. 6) TMB substrate aspirated. 7) 100 µL standard dilutions, blank, and samples added to wells (per layout). One-hour incubation (37˚C, covered). 8) Liquid removed from each well. 9) 100 µL of detection reagent A working solution added to each well. One-hour incubation (37˚C, covered).   Note. α-amylase = alpha-amylase, CRP = C-reactive protein, MUC5B/7 = mucins 5B and 7, OD = optical density, NSB = non-specific binding, TMB = tetramethylbenzidine, RA = reducing agent, BSA = bovine serum albumin, BCA = bicinchoninic acid.    20 Assay Kit Standard Protocol  α-amylase Cortisol CRP Total Protein MUC5B & MUC7 6) Plate set to mix at 500 RPM (37˚C). 7) Plate transferred to plate reader after one minute to read OD values. 8) Plate returned to mix, OD re-read after three minutes.  9) Results calculated using two OD readings and unit conversion factor, and compared to standard curve.  (Salimetrics, LLC., 2016)  5) Microplate washed four times with 1x wash buffer using a plate washer, then blotted on paper towel. 6) 200 µL of TMB substrate solution added to each well via multichannel pipette. Microplate mixed on a plate rotator at 500 RPM for 5 minutes and covered to incubate in the absence of light for 25 minutes (room temperature).  7) 50 µL stop solution added to each well via multichannel pipette. 8) Microplate mixed on a plate rotator at 500 RPM for 3 minutes (or until all of the wells turned yellow).  9) Bottom of microplate wiped and read on a plate reader at 450 nm. OD results calculated and compared to standard curve.  (Salimetrics, LLC., 2016)  7) Diluted conjugate solution immediately mixed and 150 µL added to each plate well via multichannel pipette. Microplate covered and mixed continuously on a plate rotator for two hours (room temperature).  8) Microplate washed four times with 1x wash buffer using a plate washer, then blotted on paper towel. 9) 200 µL of TMB substrate solution added to each well via multichannel pipette. Microplate covered and mixed on a plate rotator to mix continuously at 500 RPM for 30 minutes in the absence of light. 10) 50 µL stop solution added to each well via multichannel pipette. Microplate mixed on plate rotator for 3 minutes (or until all of the wells turned yellow).  11) Bottom of microplate wiped and read on a plate reader at 450 nm. OD results calculated and compared to standard curve.  (Salimetrics, LLC., 2017) 9) Microplate covered and mixed on rotator for one minute. Plate incubated for 15 minutes at 37˚C. 10) 260 µL of BCA working reagent added to each well. Plate covered and mixed for one minute, and incubated for 30 minutes at 37˚C. 11) Plate cooled to room temperature (5 minutes). 12) Standard control used as a blank. Absorbance of standards, samples, and samples controls measured at 562 nm on plate reader. 13) Average absorbance value of sample controls subtracted from the absorbance of all samples and compared to the standard curve to determine protein concentrations.  (Pierce Biotechnology, n.d.) 10) Working solution aspirated and microplate washed three times with 350 µL of 1x wash solution using a plate washer, then blotted on paper towel. Remaining wash buffer aspirated and plated blotted on paper towel. 11) 100 µL of detection reagent A working solution added to each well. Plate covered for 30-minute incubation (37˚C). 12) Working solution aspirated and microplate washed five times with 350 µL of 1x wash solution using a plate washer, then blotted on paper towel. Remaining wash buffer aspirated and plated blotted on paper towel. 13) 90 µL of substrate solution added to each well. Plate covered and incubated for 10-20 minutes at 37˚C in the absence of light. 14) 50 µL of stop solution added to each well. Gently mixed for uniform colour change (yellow). 15) Bottom of microplate wiped and read on a plate reader at 450 nm. OD results calculated and compared to standard curve.  (Cloud-Clone Corp., n.d.)  Note. α-amylase = alpha-amylase, CRP = C-reactive protein, MUC5B/7 = mucins 5B and 7, OD = optical density, NSB = non-specific binding, TMB = tetramethylbenzidine, RA = reducing agent, BSA = bovine serum albumin, BCA = bicinchoninic acid.    21 Demographics We collected demographic information from all participants, including sex, age, date of birth, type of SS, date of SS diagnosis, SS diagnostic test, other medical diagnoses, medications, and smoking history. We summarized only select demographic information using frequencies and ranges to preserve participant anonymity given the small sample.  2.6.2 Qualitative Measure  SWAL-QOL Survey Participants completed a 44-item swallowing quality of life questionnaire during or in advance of their participation appointment at UBC (SWAL-QOL, McHorney et al., 2002). The SWAL-QOL targets 10 domains (burden, eating desire, eating duration, food selection, communication, fear, mental health, social functioning, fatigue, and sleep), each of which contribute to the total SWAL-QOL score. An additional domain, dysphagia symptoms, has respondents reflect on symptom nature and frequency though does not contribute to overall scoring. As such, we summarized total SWAL-QOL scores and the symptom domain separately.   2.7 Analyses  2.7.1 Scoring and Descriptive Summaries Given our small sample size, all data were summarized using both means (standard deviations [SD]), medians (interquartile ranges [IQR]), and ranges where appropriate. Participant performance on the OSMSE-3 and CODS were calculated according to published methods (Osailan et al., 2012; St. Louis & Ruscello, 2000). For the OSMSE-3, we summarized item-based and total structural and functional deviation scores with DDK, reporting overall pass/fail results for all participants. We summarized CODS data descriptively as frequency counts for individual items, and the total scores for each group (n1, n2), and for the overall sample (N).   22 Sialometric data (weight, flow rate, and acidity) were summarized by collection method and according to group with saliva collection duration data used to calculate flow rate.  Salivary protein/hormone data were summarized by saliva collection method. Due to an anticipated small sample size, we were underpowered to conduct repeated measures analyses. Despite this, we chose to perform data correction as described below. When sample volume was insufficient to facilitate protein/hormone testing, values were replaced with the group mean value. Values that fell above the upper limit of detection were replaced with the value of this upper limit. Outliers that remained below the upper limit of detection were transformed via Winsorization and replaced with the next highest value in the group (Tukey, 1970). MUC5B and CRP values were also compared to total protein content (when not replaced with means or upper limits) to determine the specific protein content per unit of total protein. MUC5B and CRP concentrations were divided by total protein content to obtain corrected concentrations.  2.7.2 SWAL-QOL  Participants scored each SWAL-QOL item on a scale between 1 and 5 (least to most favourable). To derive each domain score, the sum of the domain-item scores was divided by the number of items within each domain (Plowman-Prine et al., 2009). The total SWAL-QOL score was determined by dividing the summed domain scores by 10 (domains). As a result, domain and total SWAL-QOL scores were expressed as a percentage, ranging from 0 (least favourable) to 100 (most favourable). We summarized the SWAL-QOL data descriptively for the 10 domain scores and total SWAL-QOL scores. These values were calculated for each group and for the overall sample. Within the domain of symptom frequency, we calculated means and standard deviations for each of the 14 items for both groups.    23 2.7.3 Exploratory Comparisons  All comparisons in this study were exploratory. When normally distributed, we compared group means of those with and without SS using an independent samples t-test. Alternatively, we compared group medians via Mann-Whitney U test for those data that were not normally distributed. We utilized Pearson product-moment correlation to analyze the relationship between salivary data (flow rate, protein/hormone concentrations) and 1) CODS total scores, and 2) SWAL-QOL data (total score, domains, symptoms). All data were analysed using IBM SPSS® Statistics 26.0 and GraphPad Prism 8.1.2 software. We set statistical significance at p < .05.     24 Chapter 3: Results 3.1 Study Feasibility 3.1.1 Participant Recruitment and Characteristics  During the three-month (13-week) recruitment period, we received a total of 14 case and six control inquiries (20 inquiries overall, Table 3.1). Of those, eight (40%) did not enrol: two (25%) did not meet inclusion criteria, and six (75%) were unable to participate due to distance from study site. The remaining twelve (N, 60%) met our inclusion criteria with all consenting and enrolling (cases [n1] = 6, controls [n2] = 6). Our approximate recruitment rate was one participant per week. Data collection occurred from one day to six weeks following initial contact and study explanation. We summarize weekly recruitment and enrolment in Table 3.2. Enrolment was discontinued after April 11, 2019 due to operational limitations.  Our convenience sample (N) was 10 females and two males with ages ranging from 31 to 68 years (y). We matched cases (n1) and controls (n2) for age (n1: 31 to 68 y; n2: 31 to 64 y) and sex with five females and one male in each group. We collected other demographic information, however did not report it to preserve participant anonymity given the small sample (Table 3.3). All cases had pSS with time since diagnosis ranging from one to 20+ years. SS diagnoses were made by physicians and methods included: extractable nuclear antibody blood testing, lip biopsy, salivary gland biopsy, and/or Schirmer’s eye test. Two participants (a matched pair) reported a remote smoking history (ceased for 10+ years).   25 Table 3.1 Participant Eligibility According to Recruitment Method  Recruitment Cases Controls Inquiries Inclusion criteria met Enrolled Inquiries Inclusion criteria met Enrolled        Posters      UBC Friedman Building* 0 0 0 0 0 0      UBC health clinicsa* 0 0 0 0 0 0      St. Paul’s Hospital* 1 1 1 0 0 0 Online      SASS webpage† and social mediab† 0 0 0 0 0 0      Si-Lab website† 0 0 0 0 0 0      Reddit pagesc† 7 4 4 0 0 0 In-Person      Word of mouth 3 1 1 6 6 6 Source unknown  3 0 0 0 0 0 Totals 14 6 6 6 6 6  Note. UBC = University of British Columbia, SASS = School of Audiology and Speech Sciences, Si-Lab = Swallowing Innovations Lab.  aUBC Health clinics included UBC Student Health Clinic, UBC Family Medical Clinic, University Village Medical Clinic, and Careville Medical Clinic. bSASS social media included Facebook and Twitter pages. cVancouver, BC and Sjogren’s Syndrome Reddit pages.  *Case recruitment poster display. †Case recruitment details posted.     26 Table 3.2 Recruitment Methods and Enrolment by Study Week  Recruitment Week   Postings  1 2 3 4 5 6 7 8 9 10 11 12 13 Jan 14-31a Feb 01-28 Mar 01-31 Apr 01-11  UBC Friedman Building*   All postings removed.  UBC health clinicsb* and St. Paul’s Hospital*  SASS webpage† and social mediac†  Reddit pagesd†  Si-Lab website Word of mouth   Total    Respondents 0 1 0 8 2 1 0 0 1 1 3 2 1 20                Cases enrolled 0 0 0 2 2 0 0 0 1 0 1 0 0 6                Controls enrolled 0 0 0 0 0 0 0 0 0 1 2 2 1 6  Note. Grey shading indicates recruitment period. UBC = University of British Columbia, SASS = School of Audiology and Speech Sciences, Si-Lab = Swallowing Innovations Lab.  aRecruitment initiated mid-January 2019, therefore no recruitment data reported for all weeks in the month of January. bUBC Health clinics included UBC Student Health Clinic, UBC Family Medical Clinic, University Village Medical Clinic, and Careville Medical Clinic. cSASS social media included Facebook and Twitter pages. dVancouver, BC and Sjogren’s Syndrome Reddit pages.  *Case recruitment poster display. †Case recruitment details posted.     27 Table 3.3 Summary of Participant Characteristics  ID Demographics                             Medical Variables  Sex  Age (years) Years post-SS diagnosis  Other diagnoses  Cases* (n = 6)   P1 M 33 6 None P2 F 31 4 Dermatological conditions, interstitial cystitis P3 F 40 1 Raynaud’s disease P4 F 40 6 Asthma, cardiac history, arthritis, Raynaud’s disease P5 F 49 21 Plantar fasciitis P6 F 68 6 None Controls (n = 6)   P7 M 35 -- None P8 F 31 -- None P9 F 37 -- None P10 F 43 -- None P11 F 49 -- Insomnia, anxiety, intermittent migraines P12 F 64 -- Grave's disease, NSTEMI  Note. ID = participant identifier, SS = Sjogren’s syndrome, NSTEMI = Non-ST-elevation myocardial infarction.  *All cases had primary SS.  3.1.2 Operational Requirements In order to conduct this study, operational requirements included: personnel, laboratory and equipment time, and associated costs. The total cost of the study was $14,900.00 and of that, $4,145.00 constituted in-kind contributions. Total costs and in-kind contributions according to category were (respectively); personnel: $7,340.00 and $2,340.00, laboratory usage: $1,350.00 (subsidized fully in-kind), and equipment: $6,210.00 and $455.00. The largest proportion of in-kind contribution was from the W-Lab and included personnel (24 hours; $1,680.00), laboratory usage (33 hours; $825.00), and equipment (12 hours, $240.00). Si-Lab in-kind contributions included personnel (33 hours; $660.00), laboratory usage (21 hours; $525.00), and equipment ($140.00). Throughout the study, personnel time totalled 81 hours and responsibilities included   28 recruitment, consenting, data collection, training, and salivary analyses. Equipment requirements included: laboratory space, computer, printer, recording device, and supplies for oral cavity examination, saliva collection, and salivary analyses. Overall equipment usage duration and laboratory time totaled 54 hours. All expenses are summarized in Appendix E.   3.2 Quantitative Measures Oral Speech Mechanism Screening Examination All participants presented with oral motor structure and function within normal limits. According to the OSMSE-3, one participant presented with structural deviations of the teeth and jaw, and reduced breath support during DDK, however was still within normal clinical limits. No participant reported signs or symptoms of swallowing difficulties. Clinical Oral Dryness Score Mean (SD) and median (IQR) overall oral dryness scores across all study participants were 1.8 (1.9) and 1.5 (3.0), respectively. Both mean (SD) and median (IQR) dryness scores for cases were numerically higher than that of controls (n1: 2.7 [2.3], 3 [5.0] versus n2: 0.8 [0.98] and 0.5 [2]), however they were not significantly different. Score ranges for cases and controls were from 0.0 to 5.0 (n1) and 0.0 to 2.0 (n2), respectively. For both groups, the most frequently scored feature was tongue adhesiveness (67% [n1] and 50% [n2]). For the remaining features, cases scored others more frequently when compared to controls: glassy mucosa (67% [n1] and 0% [n2]) and dry (no pooling of) saliva (67% [n1] and 17% [n2]). 3.2.1 Salivary Analyses Sialometry & Acidity Sialometric and salivary acidity data are summarized descriptively according to saliva collection method and group (Table 3.4). In general, overall mean/median weight and flow rate    29 Table 3.4 Sialometry and Acidity Results According to Collection Method and Group Sample Overall (N = 12)  Cases (n = 6)  Controls (n = 6)  p-valuea Range Mean (SD) Median (IQR) Range Mean (SD) Median (IQR) Range Mean (SD) Median (IQR)          Weight (g)  UWS-A 0.01-1.95 1.09 (0.57) 1.19 (0.83) 0.01-1.56 0.84 (0.65) 0.95 (1.20) 0.94-1.95 1.34 (0.38) 1.19 (0.67) 0.15 --         UWS-B 0.12-1.62 0.99 (0.57) 0.99 (1.11) 0.12-1.08 0.63 (0.35) 0.65 (0.59) 0.24-1.62 1.33 (0.54) 1.54 (0.42) -- 0.04*         SWS 0.41-1.03 0.70 (0.19) 0.65 (0.35) 0.55-1.03 0.72 (0.18) 0.68 (0.27) 0.41-0.94 0.67 (0.22) 0.62 (0.43) 0.70 --  Flow Rate (mL/min)  UWS-A 0.00-0.81 0.35 (0.26) 0.28 (0.50) 0.00-0.66 0.23 (0.24) 0.19 (0.33) 0.22-0.81 0.48 (0.24) 0.45 (0.43) 0.10 --         UWS-B 0.01-0.69 0.27 (0.22) 0.20 (0.37) 0.01-0.22 0.10 (0.08) 0.09 (0.17) 0.10-0.69 0.23 (0.19) 0.45 (0.22) 0.003* --         SWS 0.10-0.52 0.27 (0.14) 0.27 (0.26) 0.25-0.52 0.35 (0.11) 0.34 (0.17) 0.10-0.41 0.20 (0.13) 0.14 (0.22) -- 0.13  Acidity (pH value)        UWS-A 6.50-7.25 7.00 (0.24) 7.00 (0.44) 6.50-7.25 6.92 (0.30) 6.88 (0.56) 7.00-7.25 7.08 (0.13) 7.00 (0.25) -- 0.40  Note. SD = standard deviation, IQR = interquartile range, UWS-A = unstimulated whole saliva via saliva collection aid, UWS-B = unstimulated whole saliva via swab, SWS = stimulated whole saliva via swab. aFor normally distributed data, means were compared using an independent samples t-test; for non-normally distributed data, medians were compared using Mann-Whitney U test. *p < .05.   30 decreased over the appointment period. For only one collection method, UWS-B, mean flow rate differed significantly across groups (p = .003), as did median weight (p = .02). For stimulated saliva, case mean flow rates were higher relative to their unstimulated sample flow rates (SWS > UWS-A > UWS-B); however, this did not correspond with median weights. Highest median weights were noted in UWS-A. In addition, mean stimulated flow rate was higher in cases relative to controls. Comparatively, in controls, the mean flow rate decreased across the appointment period. Overall pH values (mean [SD]; median [IQR]) across all study participants were 7.00 (0.24) and 7.00 (0.44), respectively. According to group, mean (SD) and median (IQR) were: n1 - 6.92 (0.30) and 6.88 (0.56); n2 - 7.08 (0.13) and 7.00 (0.25), though did not differ significantly. Sialochemistry We describe sialochemical data according to analyte, sample, and group in Table 3.5.  Total Protein Overall total protein content (mean [SD]; median [IQR]) for each sample across all study participants was: UWS-A - 2136.6 (1179.0) and 1852.95 (1683.09), UWS-B - 1446.2 (394.0) and 962.8 (1292.0), and SWS - 1875.1 (397.6) and 1145.8 (1089.8). Across groups, mean UWS-A total protein content differed significantly (p = .003). We also observed a wider range and higher mean total protein content for cases across UWS-B and SWS, as compared to controls, though not significantly different (Table 3.5).  Alpha-amylase, Cortisol, CRP, MUC5B, and MUC7 Alpha-amylase, cortisol, CRP, and MUC5B overall mean (SD) and median (IQR) values are described in Table 3.5 according to group and collection method. When comparing cases to controls, case mean (SD) and median (IQR) concentrations for α-amylase, cortisol, and CRP   31 Table 3.5 Assay Results According to Collection Method  Sample Overall  (N = 12) Cases (n = 6)a Controls (n = 6)b p-valuec    Range Mean (SD) Median (IQR) Range Mean (SD) Median (IQR) Range Mean (SD) Median (IQR)          Total Protein (µg/mL)  UWS-A 526.69-4282.53 2136.58 (1179.02) 1852.95 (1683.09) 1518.30-4282.53 3010.03 (961.98) 3010.00 (1614.79) 526.69-1943.28 1263.14 (549.37) 1286.83 (1096.67) 0.003* --         UWS-B 2.52-5043.00 1446.23 (394.01) 962.79 (1291.96) 2.52-5043.00 2102.57 (1699.03) 2043.15 (2377.47) 218.76-1410.15 789.90 (421.71) 883.47 (683.23) 0.10 --         SWS 766.81-5131.88 1875.10 (397.57) 1145.79 (1089.83) 894.49-4237.39 2615.66 (1656.46) 2332.23 (3115.51) 766.81-1758.60 1134.55 (335.86) 1067.65 (371.49) 0.08 --  α-amylase (U/mL)  UWS-A 3.77-474.94 129.33 (122.85) 100.94 (105.10) 3.77-474.94 176.73 (161.71) 167.97 (198.54) 38.21-138.42 81.92 (40.76) 68.72 (79.42) -- 0.57         UWS-B 7.05-275.36 99.43 (85.05) 77.16 (90.01) 7.05-275.36 138.84 (106.12) 132.54 (217.51) 25.26-102.01 60.02 (30.41) 53.30 (55.35) 0.11 --         SWS 4.26-518.73 161.96 (136.98) 133.17 (139.85) 4.26-518.73 192.75 (177.56) 176.79 (231.57) 62.48-264.70 131.17 (86.47) 89.71 (161.25) 0.46 --  Note. SD = standard deviation, IQR = interquartile range, UWS-A = unstimulated whole saliva via saliva collection aid, UWS-B = unstimulated whole saliva via swab, SWS = stimulated whole saliva via swab, α-amylase = alpha-amylase, CRP = C-reactive protein, MUC5B = mucin 5B. aPrior to data correction for insufficient volume: n = 2 (UWS-A), n = 5 (UWS-B), n = 6 (SWS). bPrior to data correction for outliers: n = 4 (UWS-A) and insufficient volume: n = 5 (UWS-B), n=6 (SWS). cFor normally distributed data, means were compared using an independent samples t-test; for non-normally distributed data, medians were compared using Mann-Whitney U test. *p < .05.      32  Sample Overall (N = 12) Cases (n = 6)a Controls (n = 6)b p-valuec    Range Mean (SD) Median (IQR) Range Mean (SD) Median (IQR) Range Mean (SD) Median (IQR)          Cortisol (µg/dL)  UWS-A 0.06-0.32 0.18 (0.09) 0.17 (0.14) 0.18-0.32 0.25 (0.06) 0.25 (0.12) 0.06-0.15 0.11 (0.03) 0.11 (0.05) <.001* --         UWS-B 0.04-0.30 0.16 (0.08) 0.16 (0.12) 0.11-0.30 0.20 (0.08) 0.19 (0.16) 0.04-0.22 0.11 (0.07) 0.09 (0.13) 0.06 --         SWS 0.04-0.27 0.12 (0.07) 0.10 (0.07) 0.08-0.27 0.16 (0.07) 0.15 (0.13) 0.04-0.12 0.08 (0.78) 0.08 (0.07) 0.05 --         CRP (ng/mL)  UWS-A 0.70-21.81 7.46 (6.49) 5.78 (9.70) 8.56-21.81 12.84 (4.75) 12.27 (5.97) 0.70-3.00 2.08 (0.78) 2.17 (1.00) -- 0.002*         UWS-B 0.53-11.36 4.87 (3.53) 2.94 (5.50) 2.77-11.36 7.67 (2.77) 7.90 (2.71) 0.53-3.00 2.06 (0.94) 2.18 (1.56) -- 0.009*         SWS 0.86-13.47 4.50 (3.79) 3.03 (4.90) 3.05-13.47 7.21 (3.66) 6.97 (5.62) 0.86-3.00 1.77 (0.75) 1.64 (1.19) 0.005* --  MUC5B (ng/mL)  UWS-A 0.31-2.87 1.64 (0.93) 1.52 (1.49) 0.31-2.84 1.55 (1.08) 1.61 (2.34) 0.70-2.87 1.74 (0.85) 1.49 (1.59) 0.74 --  Note. SD = standard deviation, IQR = interquartile range, UWS-A = unstimulated whole saliva via saliva collection aid, UWS-B = unstimulated whole saliva via swab, SWS = stimulated whole saliva via swab, α-amylase = alpha-amylase, CRP = C-reactive protein, MUC5B = mucin 5B. aPrior to data correction for insufficient volume: n = 2 (UWS-A), n = 5 (UWS-B), n = 6 (SWS). bPrior to data correction for outliers: n = 4 (UWS-A) and insufficient volume: n = 5 (UWS-B), n=6 (SWS). cFor normally distributed data, means were compared using an independent samples t-test; for non-normally distributed data, medians were compared using Mann-Whitney U test. *p < .05.   33 were higher with wider ranges across all samples. We observed a significant difference between cases and controls for mean cortisol concentration (UWS-A, p < .001). For stimulated CRP, mean concentrations differed significantly across groups (p = .005), as did median concentrations for UWS-A (p = .002) and UWS-B (p = .009). When corrected in relation to total protein content, unstimulated (A) CRP concentration differed significantly across groups (p = .03). When comparing mucins between groups, MUC5B concentration did not differ significantly regardless of protein correction. Similar to α-amylase, cortisol, and CRP, cases produced a wider range of MUC5B, however MUC5B concentrations were lower as compared to controls. For all participants, MUC7 values fell below the lower limit of detection (insufficient sample volume for one case participant). As a result, MUC7 was not analyzed further. 3.3 Qualitative Measure SWAL-QOL Across all study participants, overall SWAL-QOL mean (SD) and median (IQR) total scores were 90.1 (12.8) and 94.0 (7.25), respectively. According to group, mean (SD) and median (IQR) total scores were: n1 - 83.7 (16.0) and 89.0 (18.0); n2 – 96.5 (2.4) and 95.5 (4.5). Median total scores were significantly different across groups (p = .004). When comparing domains, there were no significant differences between groups, though cases demonstrated lower median scores across all domains as compared to controls (Table 3.6). Regarding symptom frequency categories (Table 3.7), means differed significantly (p = .03) between cases and controls for: ‘thick saliva/phlegm’, ‘food sticking (throat)’, and ‘food sticking (mouth)’.      34 3.4 Exploratory Comparisons Comparing CODS and SWAL-QOL Scores to Salivary Data  We compared CODS and SWAL-QOL total scores to salivary data (flow rate, protein/ hormone concentration, Table 3.8) using a series of Pearson product-moment correlations. A significant negative correlation was determined between CODS and flow rate for both unstimulated samples (UWS-A: r = -.63, p = .03; UWS-B: r = -.60, p = .04). Alternatively, CODS positively correlated with total protein content for stimulated samples (r = .91, p < .001) and one unstimulated sample (UWS-A: r = .77, p = .003). Across collection methods, CODS positively correlated with α-amylase concentration (UWS-A: r = .83, p = .001; UWS-B: r = .81, p = .002; SWS: r = .72, p = .008). SWAL-QOL negatively correlated with total protein (SWS: r = -.81, p = .001) and α-amylase concentrations (UWS-A: r = -.83, p = .001; SWS: r = -.75, p = .005). SWAL-QOL scores did not correlate with flow rate across samples. Cortisol, CRP, and MUC5B concentrations, across samples, did not correlate with CODS nor with SWAL-QOL.  Comparing SWAL-QOL Domains and Symptoms to Salivary Data We explored correlations between SWAL-QOL domains and symptoms with salivary concentrations. Total protein, α-amylase, and MUC5B revealed significant findings. For one unstimulated collection method (A), total protein negatively correlated with the following SWAL-QOL domains: ‘burden’ (r = -.77, p = .003), ‘eating duration’ (r = -.63, p = .03), ‘symptoms’ (r = -.60, p = .04), and ‘fear’ (r = -.60, p = .04). Similar to UWS-A, stimulated (SWS) total protein negatively correlated with all domains but that of ‘eating duration’ and ‘sleep’. Domain scores did not significantly correlate with UWS-B total protein. The concentration of α-amylase negatively correlated with all domains except ‘eat duration’ for    35 Table 3.6 SWAL-QOL Scores According to Domain and Participant Group  Domain Scores (%) p-valuea  Overall (N = 12) Cases (n = 6) Controls (n = 6)    Range Median (IQR) Range Median (IQR) Range Median (IQR)         Burden  70-100 100 (15.00) 70-100 90 (22.50) 100-100 100 (0) 0.18 Eating desire  67-100 100 (0) 67-100 100 (28.50) 100-100 100 (0) 0.39 Eating duration  50-100 100 (2.000) 50-100 80.00 (50.00) 100-100 100 (0) 0.07 Symptom frequency†  57-100 96.00 (22.25) 57-100 81.50 (38.50) 91-100 98.50 (5.25) 0.07 Food selection  40-100 100 (7.50) 40-100 95.00 (30.00) 100-100 100 (0) 0.18 Communication  40-100 100 (15.00) 40-100 90.00 (30.00) 100-100 100 (0) 0.18 Fear  65-100 100 (12.50) 65-100 90.00 (23.75) 100-100 100 (0) 0.07 Mental health  72-100 100 (12.00) 72-100 92.00 (22.00) 100-100 100 (0) 0.18 Social  72-100 100 (0) 72-100 100 (13.00) 100-100 100 (0) 0.40 Fatigue  27-100 76.50 (14.00) 27-93 73.00 (31.50) 73-100 87.00 (17.25) 0.18 Sleep 20-100 75.00 (14.00) 20-100 80.00 (50.00) 60-100 75.00 (32.50) 0.82         Total Score 52-100 94.00 (7.25) 90.09 (12.79)‡ 52-94 89.00 (18.00) 83.67 (15.97)‡ 94-100 95.50 (4.50) 96.50 (2.43)‡ 0.004* --          Note. IQR = interquartile range. aFor normally distributed data, means were compared using an independent samples t-test; for non-normally distributed data, medians were compared using Mann-Whitney U test. *p < .05. † Domain score does not contribute to SWAL-QOL score. ‡Mean (SD).     36 Table 3.7 SWAL-QOL Symptom Scores According to Participant Group  SWAL-QOL Symptoms Scores† Mean Difference p-valuea Overall (N = 12) Cases (n = 6) Controls (n = 6)             Coughing 4.33 (0.89) 4.33 (1.03) 4.33 (0.82) 0 1.00 Choking (food) 4.17 (1.19) 3.50 (1.34) 4.83 (0.41) 1.33 0.06 Choking (liquids) 4.75 (0.45) 4.67 (0.52) 4.83 (0.41) 0.17 0.55 Thick saliva/phlegm 4.00 (1.48) 3.00 (1.55) 5.00 (0.00) 2.00 0.03* Gagging 4.67 (0.65) 4.33 (0.82) 5.00 (0.00) 0.67 0.10 Drooling 4.83 (0.39) 4.67 (0.52) 5.00 (0.00) 0.33 0.18 Problems chewing 4.25 (1.36) 3.50 (1.64) 5.00 (0.00) 1.50 0.08 Excess saliva/phlegm 4.67 (0.65) 4.33 (0.82) 5.00 (0.00) 0.67 0.10 Throat clearing 3.67 (1.37) 3.00 (1.55) 4.33 (0.82) 1.33 0.10 Food sticking (throat) 4.17 (1.27) 3.33 (1.34) 5.00 (0.00) 1.67 0.03* Food sticking (mouth) 4.25 (1.14) 3.50 (1.23) 5.00 (0.00) 1.50 0.03* Food/liquid out of mouth 4.75 (0.62) 4.50 (0.84) 5.00 (0.00) 0.50 0.20 Food/liquid out of nose 4.75 (0.45) 4.50 (0.55) 5.00 (0.00) 0.50 0.08 Coughing stuck food/liquid out of mouth 4.50 (1.00) 4.17 (1.33) 4.83 (0.41) 0.67 0.29  aFor normally distributed data, means were compared using an independent samples t-test; for non-normally distributed data. *p < .05. † Reported as mean (SD).  37 UWS-A, and all but ‘eat duration’, ‘symptoms’, ‘fear’, and ‘mental health’ for SWS. For UWS-B, α-amylase negatively correlated with the ‘communication’ (r = -.61, p = .04) and ‘fatigue’ (r = -.61, p = .04) domains. MUC5B (UWS-A) negatively correlated with one domain: ‘sleep’ (r = -.58, p = .047). Cortisol and CRP, regardless of collection method, were not significantly correlated to domain scores. Similar to the SWAL-QOL domains, comparisons between SWAL-QOL symptoms and salivary concentrations also resulted in significant findings for total protein and α-amylase. Total protein (UWS-A) negatively correlated with the symptoms ‘choking (food)’ (r = -.76, p = .004), ‘food sticking (throat)’ (r = -.76, p = .004), and ‘food sticking (mouth)’ (r = -.74, p = .007). All symptom categories but one (excess saliva/phlegm) correlated with stimulated total protein. Similar to total protein, α-amylase (UWS-A) negatively correlated with many symptoms: ‘choking (food)’ (r = -.65, p = .02), ‘choking (liquid)’ (r = -.64, p = .03), ‘gagging’ (r = -.70, p = .01), ‘drooling’ (r = -.75, p = .005), ‘food sticking (throat)’ (r = -.56, p = .050), and ‘coughing out stuck food/liquid’ (r = -.79, p = .002). Significant negative correlations were also observed with stimulated α-amylase (SWS) capture and the following SWAL-QOL symptoms: ‘choking (liquid)’ (r = -.73, p = .009), ‘gagging’ (r = -.60, p = .04), ‘drooling’ (r = -.66, p = .02), and ‘coughing out stuck food/liquid’ (r = -.69, p = .01). Across samples, MUC5B, CRP, and cortisol values were not significantly correlated with the 14 SWAL-QOL symptoms.            38 Table 3.8 Comparing CODS, SWAL-QOL, and Salivary Data   Note. Grey shading indicates correlation not statistically significant (p > .05). Positive (+) and negative (-) significant correlations indicate p < .05 for r > 0 and r < 0, respectively. UWS-A = unstimulated whole saliva via saliva collection aid, UWS-B = unstimulated whole saliva via swab, SWS = stimulated whole saliva via swab, α-amylase = alpha-amylase, CRP = C-reactive protein, MUC5B = mucin 5B.  aComparisons made using Pearson product-moment correlation analyses.   Comparatorsa Total Score SWAL-QOL   CODS SWAL-QOL Domains Symptoms      UWS-A       Flow Rate -  n/a n/a Total Protein +  - (4 domains) - (3 symptoms) α-amylase + - - (10 domain) - (6 symptoms) MUC5B   - (1 domain)   Cortisol     CRP      UWS-B       Flow Rate -    n/a  n/a Total Protein     α-amylase +  -  (2 domains)   Cortisol     CRP      SWS       Flow Rate    n/a  n/a Total Protein + - - (9 domains)  - (13 symptoms)  α-amylase + - - (7 domains)  - (4 symptoms)  Cortisol     CRP        39 Chapter 4: Discussion  Our study was the first to analyze the link between quantity and quality of saliva and perceptions of swallowing in those with and without SS. In doing so, we have determined that this study was feasible, while identifying significant findings across saliva composition and perceptual aspects of swallowing. In line with previous work, we identified differences in saliva volume and composition between those with and without SS. Salivary weight and flow rate differed significantly across groups for one collection method (UWS-B). Each of our measured analytes differed between groups, with significant differences in total protein and cortisol (UWS-A), and for CRP (regardless of collection method). In regard to swallowing, we found greater oral dryness and quality of life differences when comparing cases to controls. In addition, those with SS reported significant salivary symptoms related to viscosity and lubrication when compared to healthy controls. For the first time, our analyses showed significant correlations between salivary volume and composition when compared to perceptions of oral dryness, swallowing, and quality of life. Therefore, salivary biomarkers and their link to an individual’s perception of their swallowing is a valid area of investigation on a larger scale.  4.1 Study Feasibility Throughout our study, we monitored the operational requirements for study conduct. We utilized a variety of recruitment methods, some more successfully than others. The majority of case participants were recruited through social media channels with one case participant recruited via hospital advertising. In contrast, all control participants were recruited by word of mouth. We received no responses to advertisements in UBC campus clinics. Moreover, upon removal of recruitment documentation, it was revealed that it had not been displayed in many clinical sites. While posting confirmation was requested, additional follow-up should be   40 considered in our future work. Although our recruitment was successful, should a larger sample be sought, more outreach would likely be required, particularly for matched controls.  Our study was successful in part due to high recruitment rate, together with two well-appointed and complementary research laboratories. Despite a short recruitment period of 13 weeks, we obtained a high enrolment rate (60% of all inquiries), with an approximate recruitment rate of one participant per week. This rate would make a larger study of this design likely successful in the future. With respect to operational requirements, we were able to conduct much of this study utilizing existing laboratory infrastructure and equipment thereby minimizing costs. Both research laboratories provided in-kind contributions ranging from content expertise, personnel support, and equipment usage. Si-Lab is a dry lab with ample space for participant interviewing, data collection, and computing infrastructure for analyses. The W-Lab is a well-established wet lab, fully configured for large sample studies and proteomic analyses with state of the art equipment. These two labs are ideally complementary for a mixed methods study such as ours, and given their proximity, make it facile to have an efficient workflow from participant recruitment to the final analyses. Furthermore, in-house lab expertise and support, particularly at the W-Lab, also reduced study costs and time. For example, time spent with each participant and time required for assay completion was approximately half of what we anticipated.  A key component under investigation was determining the most efficient saliva capture methods given our population of interest. While saliva is relatively easy to collect for biomarker analyses when compared to other bodily fluids such as blood, studies utilizing saliva may still face logistical limitations in the areas of collection, transport, and timing. Firstly, immediately following collection, saliva should be put on ice in order to ensure the biological stability of the targeted analytes (Salimetrics, LLC, 2019). For our study, we lost six participants due to their   41 distance from the collection site. While this is not insurmountable, as many studies have collected saliva from participants remotely (Adam et al., 2011; Stroud, Chen, Doan, & Granger, 2019; Waller et al., 2016; Winchester, Sullivan, Roberts, Bryce, & Granger, 2018), geography does play a pragmatic role. Home collection requires additional equipment and expedited transport, among other things. Due to limitations, we were unable to facilitate home collection. Secondly, in order to control for normal diurnal rhythms, appointments were restricted to the late-afternoon. For these reasons, scheduling of appointments was limited to a maximum of two per day. In our study, we addressed this limitation by booking participants on weekends. For a larger study, these requirements would necessitate either more personnel to facilitate collection, flexible personnel scheduling, and/or a much longer enrolment period.  We trialed a number of different saliva collection methods with each providing different benefits in regard to significant findings and ease. With respect to saliva weight and flow rate, unstimulated collection using the swab most frequently yielded significant differences between groups. Conversely, saliva capture via saliva collection aid (SCA) generated the most significant mean differences across analytes, namely, total protein, cortisol, and CRP. Across all saliva collection devices, we collected volumes sufficient for all protein/hormone analyses for the majority of participants. For unstimulated saliva collection, 75% of our participants (n1 = 2/6; n2 = 6/6) provided sufficient volumes using the SCA and 83% (n1 = 5/6; n2 = 5/6) when using the swab. All participants were able to provide sufficient volume for stimulated collection. For some cases, SCA use proved difficult, with saliva often remaining stuck in the SCA rather than flowing through, limiting sample volume collected. This may be due to the salivary changes characteristic of SS (e.g., low flow, altered viscosity). As a result, using the SCA appeared better for the healthy individuals in our study. In the future, for those with SS, passive collection may   42 be more easily accomplished by expectoration directly into a capture tube rather than a narrow straw-like device (when not utilizing oral swabs). When resources and time allow, a practice trial across participants in order to orient them to the collection devices and process may be of benefit. Alternatively, use of sensory triggers as a means of stimulating salivation within passive collection may be warranted for populations for whom saliva production is reduced at baseline. Regardless, the utility of these collection methods is effective and in light of our findings, the method by which it is collected should be based on study objectives, design, and targeted population. For our study, we permitted saliva collection to cease according to participant comfort. While we required saliva collection to continue for a minimum of two minutes up to a maximum of five, we noted high variability in collection times. The majority of case participants provided samples for the maximum duration of five minutes regardless of collection method (UWS-A: 83% [5/6], UWS-B: 100% [6/6], SWS: 50% [3/6]). In contrast, one control utilized the maximum duration for only one sample (UWS-A). This duration variability may affect study outcomes by limiting proposed analyses. As such, all participants should attempt collection for a standard duration. This duration should be determined by investigators taking into consideration ceiling effects and swab absorbance capacity (Beltzer et al., 2010) so as to collect sufficient sample volume to facilitate all laboratory testing measures. 4.2 Salivary Biomarkers: Sialometric and Sialochemical Analyses There were some sialometric and sialochemical findings that were unexpected, which may be related to collection methods. During our collection, we noted an overall decrease in median weight and mean flow rate across unstimulated collection in all participants with the lowest flow rate noted during stimulated collection in controls. While average flow rate for   43 unstimulated samples did not fall below 0.1 mL/min to indicate hyposalivation (Humphrey & Williamson, 2001), flow rate fell at or below this cut-off for approximately half of the cases for each unstimulated saliva sample. Average and individual stimulated flow rates, across groups and participants, fell below 0.7 mL/min (Hopcraft & Tan, 2010) indicating hyposalivation. However, given that flow rate should be considered an individualized measure, suggesting comparison to one’s own baseline flow, and bearing in mind that cut-off values are specific to general populations (Humphrey & Williamson, 2001), the nature of our comparisons and our sampling order may have been a factor in these low stimulated rates.  In addition, a significant difference in total protein concentration was only observed while using the SCA. Passive collection via SCA is optimized for most salivary analytes, while swab collection is analyte specific (Salimetrics, LLC, 2018). Therefore, measurement of total protein concentration via swab may have contributed to these results being below the level of significance across groups. In general, collection device and/or order likely contributed to these findings. As a result, future feasibility studies aimed at optimizing collection methodology should ensure randomization of collection method and control for timing, among other confounds.  We observed numerous differences in sialometrics and salivary analytes when comparing those with and without SS. As with other work, we observed an inverse relationship between salivary flow rates and protein concentration (Engelen et al., 2007), specifically reduced flow and increased protein content in cases, and increased flow and reduced protein content in controls. In regard to specific analytes, we found mean α-amylase and salivary cortisol concentrations to be higher in cases than controls. Given the predominant inflammatory response in SS (Garcia-Carrasco et al., 2006), and the likely influence of environmental stressors, higher   44 cortisol and α-amylase concentrations could be considered a product of disease-related inflammation and stress (Granger et al., 2007). In relation, given the asymmetrical responsivity of α-amylase and cortisol (Adam et al., 2011; Gordis et al., 2006; 2008), within groups, mean α-amylase concentrations were high while corresponding mean cortisol concentrations were low (Granger et al., 2007). Other analytes also yielded notable findings. As a general marker of inflammation (Jonsson et al., 2007), mean CRP concentrations differed significantly across groups for all saliva samples. Given the inflammatory nature of SS, it follows that this inflammatory marker would be observed in higher levels for our cases, as compared to controls. When corrected for total protein content, CRP remained elevated. Therefore, while CRP would not be considered a biomarker specific to SS, measures of CRP concentrations in relation to the inflammatory response are significant in differentiating clinical and healthy populations while potentially confirming salivary changes.  This was the first time a study explored mucins and swallowing. Mucins are a relevant protein when considering the lubricative properties of saliva (Carpenter, 2013) and an integral component of swallowing. When investigating MUC5B and MUC7, we found somewhat ambiguous results and experienced challenges. While we did not observe a significant difference in MUC5B concentrations across groups, mean MUC5B concentrations were generally lower in cases when compared to controls, as well as when corrected for total protein. Additionally, MUC7 fell below the lower limit of detection across participants. This result may be a product of one or multiple factors, which may include the selected assay kit or our assay techniques. Specifically, our mucin ELISA kits are standardized for healthy rather than clinical populations. As such, these immunoassays have a detection range and sensitivity specific to mucin concentrations in healthy populations, which may have contributed to our findings. Should future   45 studies focus on mucins and swallowing in those with SS, we suggest a focus on the mucin composition and/or structure. For example, Western blotting with antibodies and periodic acid Schiff’s staining allow for the detection of mucin proteins and mucin glycosylation, respectively, which may then be compared to determine mucin glycosylation per unit protein (Chaudhury, et al., 2015; Chaudhury, Proctor, Karlsson, Carpenter, & Flowers, 2016). Although comparable mucin concentrations have been detected in those with SS and controls, discrepant mucin glycosylation has also been identified (Chaudhury et al., 2015; 2016). Therefore, in those with SS, rather than mucin concentration, reduced quality of mucins (Castro et al., 2013); specifically, altered mucin structure, may be a distinguishing feature in association with dry mouth and perception (Chaudhury et al., 2015; 2016), specifically as related to mucin water retention (Alliende et al., 2008). Our collection methods may have been an additional factor in mucin detectability. Our study used the SCA for the saliva sample for mucins, reducing the available volumes in some cases thereby limiting our analyses. Mucins tend not to lyse from oral swabs reducing the number of available options for saliva collection. There is however emerging evidence that suggests with some lysis agents and different swab composition, specific oral swabs may be utilized for mucin analyses (D. A. Granger, personal communication, November 16, 2018). Should additional methods be made available for saliva collection allowing for mucin testing and other proteomic analyses which target mucin composition, our understanding of mucins and perceptual aspects of swallowing may be expanded. Though not tested statistically, we observed average total protein, α-amylase, cortisol, and CRP concentrations to be higher in cases as compared to controls, while average MUC5B concentrations were lower in cases. We observed a proportional increase in some concentrations,   46 up to three-fold, in cases as compared to controls, correspondent with flow rates reduced by as much as one-third in those with SS. As such, given the challenges and delays associated with SS diagnosis, salivary protein output is worth exploring as a contributor to diagnostic methods. Timely sialochemical testing may facilitate early differential diagnosis in advance of overt patient perceptions related to oral function and sensation. 4.3 Salivary Biomarkers and Perceptions of Swallowing 4.3.1 Perceiving Oral Dryness As expected, participants with SS exhibited numerically higher oral dryness scores when compared to controls, though the differences were not statistically significant. More than half of case participants (66% [4/6]) were observed to present with three features of oral dryness: tongue adhesiveness, lack of saliva pooling (floor of the mouth), and glassy oral mucosa. While more severe than that of controls, their oral dryness was not extreme and this may relate to timing of SS diagnosis. Nearly all participants with SS were diagnosed with the disease recently (six years or less) and as such, are less likely to have advanced forms of the disease and in theory, less progression of salivary gland degradation. Half of control participants (50% [3/6]) also presented with tongue adhesiveness. Given that they did not frequently present with other oral dryness characteristics, this finding is more likely circumstantial given the pre-saliva collection fasting. However, the recruitment of all control participants via word of mouth may have introduced bias. Although not diagnosed with SS, our controls may have been interested in the study due to experiencing dry mouth and the like; likewise, case participants experiencing more severe oral dryness may have been more interested in participating than those with an SS diagnosis, but lesser xerostomia. Despite this potential bias, participants were still within normal clinical limits and we were able to differentiate between the two groups. Across all participants, oral dryness   47 scores negatively correlated with unstimulated collection flow rates. This corresponds with clinical findings of more severe oral dryness associated with unstimulated, and concurrently reduced, salivary flow rates. We were the first to explore connections between perceptions related to swallowing and salivary biomarkers in SS using a matched design. To explore this comparison, we chose a variety of analytes for two reasons: 1) our aim for future studies was to uncover the relation between biomarkers understood to be theoretically influential in swallowing and 2) due to the novel area of inquiry, it was difficult to determine which biomarkers would be influential a priori. In regard to oral dryness, when comparing CODS to salivary analytes, we observed a positive correlation with total protein content in all but one collection method (UWS-B), and a positive correlation of α-amylase concentration across saliva samples. This finding is supported as oral dryness is often due to reduced salivary flow and reduced salivary flow rate leads to higher protein concentration (Engelen et al., 2007), which was perceived by the participants in our study. In contrast, oral dryness did not correlate with cortisol, CRP, and MUC5B concentrations despite our observation of higher CRP concentrations in cases as compared to controls. Given that CRP is an inflammatory marker (Jonsson et al., 2007), it is likely unrelated to swallowing and oral dryness. Similarly, we observed no correlation between cortisol concentration and the CODS despite the inverse relation of salivary cortisol and α-amylase concentration (Adam et al., 2011; Gordis et al., 2006; 2008). This too is supported given that cortisol plays no theoretical role in swallowing. This confirms our findings that α-amylase is a marker of saliva quality and oral dryness, independent of cortisol, underscoring the value of α-amylase in salivary analyses related to swallowing. This has been supported in other work in healthy participants when investigating the relation of α-amylase with oral perception (Ong,   48 Steele, & Duizer, 2018). Although mucins are also relevant for swallowing given their role in lubrication, our study did not reveal significant findings for dryness comparison. While our mucin concentrations may not have correlated, in the future, more specific analyses of mucin quality may be a more suitable indicator and area of inquiry in relation to oral dryness. 4.3.2 Quality of Life as it Relates to Swallowing According to the SWAL-QOL, those with SS reported disease burden and reduced life quality related to swallowing when compared to controls. Specific to dysphagia symptoms, those with SS more frequently reported symptoms such as ‘thick saliva/phlegm’, ‘food sticking (throat)’, and ‘food sticking (mouth)’. Our findings confirm the likely relation of salivary lubricative properties (saliva composition) with that of symptoms and disease related changes. With lower MUC5B concentrations detected on average in cases, the lubricative quality of saliva is likely to be reduced. As the lubricative role of mucins is specific to the oral preparation of food and swallowing (Carpenter, 2013; Dawes et al., 2015), it follows that case participants would report more viscous saliva, reduced salivary lubrication, and globus sensation. Similarly, case participant reports of food sticking in the oral cavity and perceptions related to salivary viscosity (Engelen et al., 2003) may also be attributed to the higher mean α-amylase concentrations due to the digestive and lubricative properties of this protein, and its role in oral cavity clearance (Aguirre et al., 1989; Carpenter, 2013). When comparing SWAL-QOL scores to salivary analytes, total scores negatively correlated with stimulated and unstimulated total protein (SWS) and α-amylase (UWS-A, SWS). This finding is likely due to concentrated protein effects. Those with SS reported lower SWAL-QOL total scores, indicative of reduced quality of life, and had higher protein concentration likely due to hyposalivation and dilution effects. The opposite, higher SWAL-QOL scores (lesser impacts to quality of life) and lower protein   49 concentration was observed for our controls. This finding however was not consistent across saliva samples. Given that we only observed this correlation for both proteins with stimulated samples, the collection method and its timing need be considered for future studies. It could be the case that greater total protein and α-amylase concentration is due to stimulation effects and, more specifically, from increased activity of the parotid glands, which predominantly secrete α-amylase (Carpenter, 2013).  When comparing salivary biomarker concentrations to SWAL-QOL domains, no consistent pattern emerged regarding domain correlates, however a consistently inverse relation was noted. In general, all significant correlations were negative, indicating an inverse relation between the salivary concentrations and quality of life, or alternatively, greater disease burden/lower life quality the thicker, less desirable saliva composition. Furthermore, saliva collection timing, method, and/or device did appear to play a role. Unstimulated saliva collected by swab (UWS-B) was the least likely to yield significant findings when compared to other methods (UWS-A and SWS). Specifically, α-amylase (all samples), total protein (SWS), and MUC5B (UWS-A) concentration were all negatively correlated to most SWAL-QOL domains. For those comparisons yielding correlations to fewer domains, total protein concentration (UWS-A), was negatively correlated with four domains (‘burden’, ‘eating duration’, ‘symptoms’, and ‘fear’), all of which overtly relate to personal perceptions specific to impacts on deglutition. As such, these correlation effects could be considered to follow from concentrated protein effects and therefore hyposalivation. In contrast, α-amylase concentration negatively correlated with two domains (‘communication’ and ‘fatigue’) for an unstimulated sample (UWS-B). Further exploration of such a finding is merited, particularly considering that these domains correlated with α-amylase concentration across samples. The lubricative properties of α-amylase (Aguirre   50 et al., 1989) may be a factor in perceived communication- and fatigue-related difficulties. Finally, a negative correlation between MUC5B concentration and the ‘sleep’ domain may be attributed to diurnal rhythms and concentrated protein effects. With low flow rates during sleep (Dawes, 2008; Turner & Ship, 2007), it would follow that mucin content would be higher; however, we should then expect to observe this correlation effect between the sleep domain and remaining analytes. Mucin quality, rather than concentration, warrants further consideration in this regard. Overall, a study with a larger sample and a targeted collection method may uncover more consistent and specific patterns in regard to particular domains and corresponding salivary biomarkers. When investigating specific dysphagia symptoms and saliva concentrations, a similar pattern to that of oral dryness and other salivary comparisons emerged. Significant findings align symptoms specific to the lubricative properties of saliva with their (theoretically) corresponding analytes, specifically, total protein and α-amylase. Across unstimulated (UWS-A) and stimulated samples, total protein and α-amylase concentrations negatively correlated with multiple SWAL-QOL symptoms, three of which overlapped (choking and food sticking in both throat and mouth). Total protein negatively correlated with all but the symptom of ‘excess saliva/phlegm’. Bearing in mind the hyposalivation that is central to this study and the clinical population of interest, this is a crucial finding. Excess secretions are not likely to be reported by those with SS, nor those of typical health, and would therefore not be associated with protein concentrations. Other symptom-concentration correlations included: ‘choking [liquid]’, ‘gagging’, ‘drooling’, ‘coughing out stuck food/liquid’ with α-amylase (UWS-A, SWS). In addition, significant correlations were more frequently observed for those analytes collected by SCA and stimulation. Again, we are unable to determine whether this is due to saliva collection ineffectiveness for our   51 study objectives or collection order and/or timing. Similar to the oral dryness correlations, cortisol and CRP concentrations across saliva samples did not significantly correlate with SWAL-QOL data. This is again attributable to the theoretical functions of CRP and cortisol and their lack of relevance to swallowing. 4.4 Limitations  This feasibility study was exploratory in nature with a primary objective to determine ideal and efficient methods of measurement across multiple parameters. As a result, our objectives were focused more generally on study conduct and utility (e.g., which unstimulated saliva collection method produced the most saliva across participant groups) rather than specific comparisons. So while we were successful in the execution of our proposed methods while identifying challenges with some collection approaches, our limited study scope did not afford us the ability to make definitive conclusions regarding salivary biomarkers and swallowing perception. Given its feasibility focus, other study limitations also included a short duration while employing a variety of saliva collection methods and measures. Our study duration was constrained by academic schedules, resulting in a short 13-week recruitment period and therefore a small sample. In addition, all participants with SS presented with the primary variant. We were able to conduct descriptive analyses, however other statistical comparisons were limited. Together, these factors limited our generalizability to the broader SS and healthy populations. Other aspects of study conduct may also limit our findings while introducing bias. Specifically, recruitment of all participants occurred in one geographic location with control recruitment occurring solely via word of mouth. This could lead to a potentially biased sample and as a result, may impact our findings reducing our ability to show differences between groups in addition to lack of representation of the whole population. In addition, our saliva collection   52 methods may have affected our results. We did not randomize the order of saliva collection and also did not collect the saliva for a specific time period (we allotted a range for saliva collection duration). Lack of control of these factors may have introduced order and ceiling effects, respectively, limiting the accuracy of our salivary findings. Nonetheless, given our outcomes, we were able to streamline study design and provide information in order to calculate sample size estimates and conduct power calculations. 4.5 Clinical Implications and Future Directions  Our observations have confirmed the correlation of saliva composition with perception in both disease and healthy conditions, as well as increased burden and dysphagia symptoms in those with SS. Should these findings be supported in future work with larger samples, these may provide prospective contribution to early treatment and management of swallowing disorders. Expanding this research could facilitate identification of SS-specific salivary biomarkers and their application to disordered swallowing and differential diagnosis. Early diagnosis may also be facilitated by explicit determination of SS-specific salivary changes (volume and composition) and impacts to swallowing (function and perception). Moreover, comprehensive knowledge of salivary changes in SS would facilitate customized treatment strategies. A link between salivary biomarkers and swallowing disorders would allow for application of these methods to other patient populations experiencing salivary changes and concomitant dysphagia, thereby supporting the clinical understanding of altered saliva on swallowing. Furthermore, the means by which dysphagia is treated may be expanded to facilitate patient-centered treatment approaches generated in view of perceptions and quality of life. Given our study’s clinical focus, our findings may also extend to those with other autoimmune or chronic illnesses and patient populations experiencing dry mouth and swallowing dysfunction. For example, extending such   53 analyses to the unique experience of those with head and neck cancers and subsequent xerostomia and dysphagia, particularly as related to radiation therapy, may be worthwhile in view of both quality of life and quality of survival. Moving forward, future work should incorporate more targeted recruitment methods, with a focus on social media and word of mouth, with more outreach indicated for larger sample sizes from multiple locations to reduce potential bias. Revision to saliva collection methods by way of facilitating home collection by participants may also facilitate recruitment and eliminate geographic restrictions. If comparing saliva collection methods, collection randomization, while controlling collection timing, should be central to future research. The CODS and SWAL-QOL were effective for our study design, and would be useful in future work. Despite this, incorporation of a semi-structured qualitative interview with thorough thematic analysis would likely facilitate identification of richer relationships between salivary and perceptual data and enhanced understanding of patient experiences. Additionally, understanding the link between swallowing and/or laryngeal physiology, salivary biomarkers, and perception is important. Incorporation of an instrumental assessment of swallow physiology would allow for comparison to swallow perceptions, as well as to distinguish salivary impacts specific to both swallow function and perception. Ideally, fiberoptic endoscopic evaluation of swallowing would afford an assessment of both swallowing and laryngeal physiology as well as an ability to review the mucosal lining of the upper aerodigestive tract. This in conjunction with stroboscopy would help to determine the effect (if any) of salivary changes on the upper airway for other populations (e.g., professional voice users). Future work should focus on salivary total protein and α-amylase concentrations, as well as investigation of mucin quality/structure rather than cortisol and CRP. Collection method may have also be a factor in our findings. Therefore, conducting repeated   54 measures or MANOVA analyses would be an effective measure to determine the effects of both collection method and group.  4.6 Conclusion Our research has expanded on investigations of salivary volume (Gonzalez et al., 2014; Hughes et al., 1987; Pijpe et al., 2007; Rhodus et al., 1995a; 1995b) and the proposed role of salivary composition (Engelen et al., 2007; Kalk et al., 2001; 2002; Mathews et al., 2008; Rogus-Pulia & Logemann, 2011; Zussman et al., 2007) in relation to swallowing in SS. Furthermore, our study was novel in combining analyses of saliva quantity and quality and perceptions of swallowing in those with and without SS. Our methodological approach facilitated examination of swallowing in terms of both function and perception, incorporating patient experiences and evaluation of life quality. Our findings of salivary differences in volume and composition, and associations to oral dryness, swallowing, and quality of life are a framework on which to build upon with respect to salivary analyses within and beyond SS. Though exploratory, this study facilitated more precise operationalization of saliva collection in healthy controls and a clinical population, with these methods applicable to future studies and other patient populations. What is more, a comprehensive understanding of salivary changes and swallowing perception in SS allows for expanded study of the disease itself, and of associated dysphagia. 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In the last month how often have you experiences each of the symptoms below.All of thetimeMost ofthe timeSome ofthe timeA little ofthe timeNone ofthe timeFeel weak 1 2 3 4 5Thank you for your help in taking part in this survey!  71     IMPORTANT NOTE: We understand that you may have a number of physical problems.Sometimes it is hard to separate these from swallowing difficulties, but we hope that youcan do your best to concentrate only on your swallowing problem.  Thank you for yourefforts in completing this questionnaire.1. Below are some general statements that people with swallowing problems might mention. In the last month, how true have the following statements been for you.(circle one number on each line)Very muchtrueQuite a bittrueSomewhattrueA littletrueNot atall trueDealing with myswallowing problem isvery difficult.1 2 3 4 5My swallowing problem isa major distraction in mylife.1 2 3 4 52. Below are aspects of day-to-day eating that people with swallowing problemssometimes talk about. In the last month, how true have the following statements been for you?(circle one number on each line)Very muchtrueQuite a bittrueSomewhattrueA littletrueNot atall trueMost days, I don’t care if Ieat or not.1 2 3 4 5It takes me longer to eatthan other people.1 2 3 4 5I’m rarely hungryanymore.1 2 3 4 5It takes me forever to eata meal.1 2 3 4 5I don’t enjoy eatinganymore.1 2 3 4 5  72     3. Below are some physical problems that people with swallowing problemssometimes experience. In the last month, how often you have experienced eachproblem as a result of your swallowing problem?(circle one number on each line)AlmostalwaysOften Sometimes HardlyeverNeverCoughing 1 2 3 4 5Choking when you eat food 1 2 3 4 5Choking when you takeliquids1 2 3 4 5Having thick saliva or phlegm 1 2 3 4 5Gagging 1 2 3 4 5Drooling 1 2 3 4 5Problems chewing 1 2 3 4 5Having excess saliva orphlegm1 2 3 4 5Having to clear your throat 1 2 3 4 5Food sticking in your throat 1 2 3 4 5Food sticking in your mouth 1 2 3 4 5Food or liquid dribbling out ofyour mouth 1 2 3 4 5Food or liquid coming outyour nose 1 2 3 4 5Coughing food or liquid out ofyour mouth when it gets stuck 1 2 3 4 54. Next, please answer a few questions about how your swallowing problem hasaffected your diet and eating in the last month.(circle one number on each line)StronglyagreeAgree Uncertain Disagree StronglydisagreeFiguring out what I can and can’teat is a problem for me.1 2 3 4 5It is difficult to find foods that Iboth like and can eat.1 2 3 4 5  73     5. In the last month, how often have the following statements about communicationapplied to you because of your swallowing problem?(circle one number on each line)All ofthe timeMost ofthe timeSome ofthe timeA little ofthe timeNone ofthe timePeople have a hard timeunderstanding me. 1 2 3 4 5It’s been difficult for me tospeak clearly. 1 2 3 4 56. Below are some concerns that people with swallowing problems sometimes mention. In the last month, how often have you experienced each feeling?(circle one number on each line)AlmostalwaysOften Sometimes HardlyeverNeverI fear I may start choking when Ieat food.1 2 3 4 5I worry about getting pneumonia. 1 2 3 4 5I am afraid of choking when I drinkliquids. 1 2 3 4 5I never know when I am going tochoke.1 2 3 4 57. In the last month, how often have the following statements been true for you becauseof your swallowing problem?(circle one number on each line)AlwaystrueOftentrueSometimestrueHardlyever trueNevertrueMy swallowing problemdepresses me.1 2 3 4 5Having to be so careful whenI eat or drink annoys me.1 2 3 4 5I’ve been discouraged by myswallowing problem.1 2 3 4 5My swallowing problemfrustrates me.1 2 3 4 5I get impatient dealing withmy swallowing problem.1 2 3 4 5  74      8. Think about your social life in the last month. How strongly would you agree ordisagree with the following statements?(circle one number on each line)StronglyagreeAgree Uncertain Disagree StronglydisagreeI do not go out to eat becauseof my swallowing problem.1 2 3 4 5My swallowing problem makesit hard to have a social life.1 2 3 4 5My usual work or leisureactivities have changedbecause of my swallowingproblem.1 2 3 4 5Social gatherings (like holidaysor get-togethers) are notenjoyable because of myswallowing problem.1 2 3 4 5My role with family and friendshas changed because of myswallowing problem.1 2 3 4 59. In the last month, how often have you experienced each of the following physical symptoms?(circle one number on each line)All ofthe timeMost ofthe timeSome ofthe timeA little ofthe timeNone ofthe timeFeel weak? 1 2 3 4 5Have trouble falling asleep? 1 2 3 4 5Feel tired? 1 2 3 4 5Have trouble staying asleep? 1 2 3 4 5Feel exhausted? 1 2 3 4 5  75 Appendix C: Recruitment Posters    Version: November 5, 2018  Page 1 of 1 PARTICIPANTS NEEDED FOR SJOGREN’S SYNDROME RESEARCH  We are looking for participants to take part in a study on salivation and swallowing in Sjogren’s syndrome.             What’s involved? As a participant in this study, you would be asked to: 1) Provide samples of your saliva. 2) Participate in an interview about your experience with Sjogren’s syndrome.  Your participation is entirely voluntary and would take approximately two hours.  What are the benefits of participating? By participating in this study, you would help us to better understand, manage, and treat salivary and swallowing changes associated with Sjogren’s syndrome. We hope that the information learned from this study can be used to benefit individuals with Sjogren’s syndrome.   In appreciation of your time, remuneration will be provided for your participation.   This study is supervised by: Dr. Stacey Skoretz  (sskoretz@audiospeech.ubc.ca; 604-822-5482) School of Audiology and Speech Sciences, Faculty of Medicine, University of British Columbia  This study has been approved by the University of British Columbia Clinical Research Ethics Board.  Who can participate?  We are looking to recruit adults who are:  • Diagnosed with primary or secondary Sjogren’s syndrome • 18 years of age or older • Able to communicate fluently in English (written and oral) • Without a recent smoking history, prior history of radiotherapy or surgery for head and neck cancer, neurological and/or neurodegenerative disease, or currently use medications which result in reduced salivation.  To learn more about, or to participate in this study, please contact: Veronica Letawsky v.letawsky@alumni.ubc.ca M.Sc. Speech-Language Pathology Candidate    76      Version: November 5, 2018  Page 1 of 1  HEALTHY VOLUNTEERS NEEDED FOR RESEARCH STUDY  We are looking for healthy individuals to take part in a research study which will compare saliva and swallowing of healthy volunteers to those who have Sjogren’s syndrome.             What’s involved? As a participant in this study, you would be asked to:  1) Provide samples of your saliva. 2) Participate in an interview about oral symptoms.  Your participation is entirely voluntary and would take approximately two hours.  What are the benefits of participating? By participating in this study, you would help us to better understand salivary and swallowing changes associated with Sjogren’s syndrome.    In appreciation of your time, remuneration will be provided for your participation.   This study is supervised by: Dr. Stacey Skoretz  (sskoretz@audiospeech.ubc.ca; 604-822-5482) School of Audiology and Speech Sciences, Faculty of Medicine, University of British Columbia  This study has been approved by the University of British Columbia Clinical Research Ethics Board.  Who can participate?  We are looking to recruit adults who are:  • Healthy with no acute or chronic health conditions • 18 years of age or older • Able to communicate fluently in English (written and oral) • Have no recent smoking history, prior history of radiotherapy or surgery for head and neck cancer, neurological and/or neurodegenerative disease, or currently use medications which result in reduced salivation.  To learn more about, or to participate in this study, please contact: Veronica Letawsky v.letawsky@alumni.ubc.ca M.Sc. Speech-Language Pathology Candidate    77 Appendix D: Consent Forms    Version: January 28, 2019  Page 1 of 6 H18-02328                                                        Participant Information and Consent Form   Saliva Production and Composition and the Perception of Swallowing Function in Sjogren’s Syndrome   Principal Investigators: Dr. Stacey Skoretz Assistant Professor School of Audiology and Speech Sciences Faculty of Medicine The University of British Columbia 421-2177 Wesbrook Mall Vancouver, BC V6T 1Z3 T: 604.822.5482   sskoretz@audiospeech.ubc.ca   Co-investigators: Dr. Camilla Dawson Clinical Lead Speech and Language Therapist University Hospitals Birmingham NHS Foundation Trust Visiting Scientist, University of British Columbia   Dr. Caroline Nguyen Assistant Professor Department of Oral Health Sciences Faculty of Dentistry | University of British Columbia  Dr. Joanne Weinberg Professor and Distinguished University Scholar, Emerita Department of Cellular and Physiological Sciences University of British Columbia   Veronica Letawsky, B.Sc. M.Sc. Speech-Language Pathology Candidate School of Audiology and Speech Sciences Faculty of Medicine | University of British Columbia v.letawsky@alumni.ubc.ca   Invitation You are invited to take part in a research study that is being done in the Swallowing Innovations Lab at the School of Audiology and Speech Sciences at the University of British Columbia. You are eligible to participate in this study because you have been diagnosed with either primary or secondary Sjogren’s syndrome.    78         Version: January 28, 2019  Page 2 of 6 H18-02328  Your Participation is Voluntary Your participation is entirely voluntary. You have the right to refuse to participate in this study. If you decide to participate, you may still choose to withdraw from the study at any time without any negative consequences.  Before you decide, it is important for you to understand what the research involves. This consent form will tell you about the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. Your involvement in the study is in addition to the care you would otherwise receive from medical professionals, as related to your diagnosis. The information acquired during the study is meant to provide new information that may help others who share your diagnosis in the future. The researchers have a duty of care to all participants and will inform you of any information that may affect your willingness to remain in the study.  If you wish to participate in this study, you will be asked to sign this form.    Please take time to read the following information carefully and, if you wish, discuss it with your family, friends, and doctor before you decide.  Who is Conducting this Study? This study is being conducted by Dr. Stacey Skoretz at the University of British Columbia. The study is not receiving funds from an external agency or sponsor.  Background  Individuals diagnosed with Sjogren’s syndrome are likely to experience changes in their saliva, including changes in both the amount and composition of saliva. Along with the symptom of dry mouth, those with Sjogren’s syndrome may experience symptoms related to swallowing function. There is potential for salivary changes to result in difficulties swallowing, which may impact other aspects of life.  What is the Purpose of this Study? This study aims to identify differences in the quantity and composition of saliva produced in those with Sjogren’s syndrome by comparing saliva samples to those of healthy individuals. In addition, we intend to explore the potential effects of changes in saliva on perceptions of swallowing. This information may help to inform future treatment methods for both swallowing dysfunction and the symptom of dry mouth. The results of this study may be used as a guide for larger studies, although there is no guarantee that they will be conducted.  A total of 40 participants will be included in this study at the University of British Columbia.  Who Can Participate in this Study? You may be able to participate in this study if you are 18 years or older, have been diagnosed with either primary or secondary Sjogren’s syndrome, and have the ability to communicate fluently in English.   Who Should not Participate in this Study? You cannot participate in this study if you have a recent smoking history, prior history of radiotherapy or surgery for head and neck cancer, neurological and/or neurodegenerative disease, or currently use medications which result in reduced salivation.    79         Version: January 28, 2019  Page 3 of 6 H18-02328  What Does the Study Involve?  If you agree to participate in this study and sign this form, your saliva will be collected by trained researchers using methods described below. You will also participate in an interview regarding salivation and swallowing, as related to Sjogren’s syndrome.  Oral mechanism examination: Prior to saliva a collection, a trained researcher will conduct an examination of your oral cavity, including both the appearance of oral structures and their function. This is estimated to require approximately 10 minutes of your time.  Assessment of oral dryness: Your oral cavity will also be assessed to determine the level of dryness. This will include assessment of the appearance of your saliva, and the appearance and adhesiveness of your oral structures. This is estimated to require approximately 5 minutes of your time.  Saliva collection: Your saliva will be collected in three different ways. You will be requested to refrain from eating, smoking, chewing gum, brushing teeth, using dental floss, and drinking anything but water for 1.5 hours prior to saliva collection (consumption of tap water allowed up to 15 minutes prior to collection). If you utilize oral moisturizers, saliva substitutes, or salivary replacement medications, you will be asked to consult with your physician and, if permitted, requested to temporarily discontinue the use of these products 48 hours prior to saliva collection. Saliva will be collected while you are sitting upright and refraining from speaking and swallowing. This is estimated to require approximately 30 minutes of your time.  Beginning with the collection of unstimulated saliva, you will be requested to expectorate all fluid within your mouth through a straw-like saliva collection aid into a tube every 30 seconds for a total duration of 2 to 5 minutes. Following a two-minute break, you will be requested to place an oral swab under your tongue for a total duration of 2 to 5 minutes, after which the swab will be placed into a storage tube. Stimulated saliva will then be collected following another two-minute break. You will be requested to chew on a second oral swab for a total duration of 2 to 5 minutes, after which the swab will be placed into a second storage tube.   All saliva capture tubes will be immediately stored on ice in the Swallowing Innovations Lab. Following which, they will be transported in a cooler to the Weinberg Lab at UBC, and frozen until subsequent analyses are completed. The samples will be analysed to determine saliva quantity and quality using a variety of tests including assays. Saliva samples will be identified by your study participation number and not linked to your name or personal information. Samples will be stored until our data collection is completed or February 2019, whichever comes first. Following which, samples will be destroyed.  Interview: Following saliva collection, you will be asked some questions regarding your experience with Sjogren’s syndrome. These questions will be focused on salivation, swallowing, and other impacts to the quality of your life. This is estimated to require approximately 60 minutes of your time.  The interview will be audio recorded and then transcribed. Your information will be stored using a unique study identifier and without using your name, so none of your information will be identifiable.  All data pertaining to this study will be stored in a locked facility, only accessible by study personnel.  Quality of life questionnaire: You will also be asked to complete a 44-item questionnaire regarding your feelings toward swallowing, your swallowing symptoms, and related impacts to your quality of life. This is estimated to require approximately 10 minutes of your time. This questionnaire will be   80         Version: January 28, 2019  Page 4 of 6 H18-02328  provided to you upon enrolling in the study and can be completed in advance or on the day of your study participation at UBC.  What are the Possible Harms and Discomforts? You may experience some mild physical discomfort during the collection of saliva as related to the symptom of dry mouth, limitations to oral intake prior to saliva collection, and the need to refrain from speaking and swallowing for the duration of saliva collection. You may experience some emotional discomfort during the interview while expressing your perceptions of the impacts of Sjogren’s syndrome on salivation, swallowing, and other aspects of life. You will have the option to take breaks and you will not have to answer any questions that you are uncomfortable with.  What are the potential Benefits of Participating? There may be no direct benefit to you from taking part in this study. However, the information gathered from the assessments may help researchers and healthcare professionals to better understand and manage salivary and swallowing changes associated with Sjogren’s syndrome. We hope that the information learned from this study can be used in the future to benefit other people with Sjogren’s syndrome.    What are the Alternatives to Participating in this Study?  If you decide not to give consent for participation in this study your decision will in no way affect the current management from your healthcare providers or the quality of care that is provided.  What if New Information Becomes Available that May Affect My Decision to Participate? You will be advised of any new information that becomes available that may affect your willingness to remain in this study.  What Happens if I Decide to Withdraw my Consent to Participate?  You may withdraw from this study at any time without giving reasons. If you choose to enter the study and then decide to withdraw at a later time, you have the right to request the withdrawal of your information collected during the study. This request will be respected to the extent possible. Please note however that there may be exceptions where the data will not be able to be withdrawn, for example, where the data is no longer identifiable (meaning it cannot be linked in any way back to your identity) or where the data has been merged with other data. If you would like to request the withdrawal of your data, please let your study doctor know. If your participation in this study includes enrolling in any optional studies, you will be asked whether you wish to withdraw from these as well.  Will my Taking Part in this Study be Kept Confidential?  Your confidentiality will be respected. However, research records and health or other source records identifying you may be inspected in the presence of the Investigator or his designate by representatives of the University of British Columbia Clinical Research Ethics Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law. Deidentified data gathered during this study will be used in research publications in medical journals and/or scientific presentations. Study data will be kept for 25 years, as per national and institutional recommendations, and then destroyed.  You will be assigned a unique study number as a participant in this study. This number will not include any personal information that could identify you (e.g., it will not include your Personal Health Number,   81         Version: January 28, 2019  Page 5 of 6 H18-02328  SIN, or your initials, etc.). Only this number will be used on any research-related information collected during the course of this study, so that your identity (i.e. your name or any other information that could identify you) as a participant in this study will be kept confidential. Information that contains your identity will remain only with the Principal Investigator and/or designate. The list that matches your name to the unique study number that is used on your research-related information will not be removed or released without your consent unless required by law.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information. Further details about these laws are available on request to your study doctor.   What happens if something goes wrong? By signing this form, you do not give up any of your legal rights and you do not release the study doctor, participating institutions, or anyone else from their legal and professional duties. If you become ill or physically injured as a result of participation in this study, medical treatment will be provided at no additional cost to you. The costs of your medical treatment will be paid by your provincial medical plan.  What Will the Study Cost Me?  You will not incur any personal expenses as a result of partaking in this study. Following your participation in the study, you will be compensated $20.  Who do I Contact if I Have Questions About the Study During My Participation? If you have any questions or desire further information about this study before or during participation, or if you experience any adverse effects, you can contact Dr. Stacey Skoretz at 604-822-5482.  Who Do I contact if I Have Concerns About My Rights as a Study Participant? If you have any concerns or complaints about your rights as a research participant and/or your experiences while participating in this study, contact the Research Participant Complaint Line in the University of British Columbia Office of Research Ethics by e-mail at  RSIL@ors.ubc.ca or by phone at 604-822-8598 (Toll Free: 1-877-822-8598). Please reference the study number [H18-02328] when calling so the Complaint Line staff can better assist you.      82         Version: January 28, 2019  Page 6 of 6 H18-02328  CONSENT TO PARTICIPATE  Saliva Production and Composition and the Perception of Swallowing Function in Sjogren’s Syndrome  My signature on this consent form means: ▪ I have read and understood the participant information and consent form.  ▪ I have had the opportunity to ask questions and have had satisfactory responses to my questions.  ▪ I understand that participation in this study is voluntary ▪ I understand that I am completely free at any time to refuse participation or to withdraw the participant from this study at any time, and that this will not change the quality of care that he or she receive. ▪ I authorize access to my health record as described in this consent form.  ▪ I understand that I am not waiving any of my legal rights as a result of signing this consent form.  I will receive a signed copy of this consent form for my own records.  I consent to participate in this study.    _________________________________________________________________________________  Printed name of participant     _________________________________________________________________________________  Participant’s        Date Signature     _________________________________________________________________________________  Signature of Person   Printed name       Role   Date Obtaining Consent      83      Version: January 28, 2019  Page 1 of 6 H18-02328                                                       Participant Information and Consent Form   Saliva Production and Composition and the Perception of Swallowing Function in Sjogren’s Syndrome   Principal Investigators: Dr. Stacey Skoretz Assistant Professor School of Audiology and Speech Sciences Faculty of Medicine The University of British Columbia 421-2177 Wesbrook Mall Vancouver, BC V6T 1Z3 T: 604.822.5482   sskoretz@audiospeech.ubc.ca   Co-investigators: Dr. Camilla Dawson Clinical Lead Speech and Language Therapist University Hospitals Birmingham NHS Foundation Trust Visiting Scientist, University of British Columbia   Dr. Caroline Nguyen Assistant Professor Department of Oral Health Sciences Faculty of Dentistry | University of British Columbia  Dr. Joanne Weinberg Professor and Distinguished University Scholar, Emerita Department of Cellular and Physiological Sciences University of British Columbia  Veronica Letawsky, B.Sc. M.Sc. Speech-Language Pathology Candidate School of Audiology and Speech Sciences Faculty of Medicine | University of British Columbia v.letawsky@alumni.ubc.ca   Invitation You are invited to take part in a research study that is being done in the Swallowing Innovations Lab at the School of Audiology and Speech Sciences at the University of British Columbia. You are eligible to participate in this study because you have not been diagnosed with Sjogren’s syndrome.    84         Version: January 28, 2019  Page 2 of 6 H18-02328  Your Participation is Voluntary Your participation is entirely voluntary. You have the right to refuse to participate in this study. If you decide to participate, you may still choose to withdraw from the study at any time without any negative consequences.  Before you decide, it is important for you to understand what the research involves. This consent form will tell you about the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. Your involvement in the study is in addition to the care you would otherwise receive from medical professionals, as related to your diagnosis. The information acquired during the study is meant to provide new information that may help others who share your diagnosis in the future. The researchers have a duty of care to all participants and will inform you of any information that may affect your willingness to remain in the study.  If you wish to participate in this study, you will be asked to sign this form.    Please take time to read the following information carefully and, if you wish, discuss it with your family, friends, and doctor before you decide.  Who is Conducting this Study? This study is being conducted by Dr. Stacey Skoretz at the University of British Columbia. The study is not receiving funds from an external agency or sponsor.  Background  Individuals diagnosed with Sjogren’s syndrome are likely to experience changes in their saliva, including changes in both the amount and composition of saliva. Along with the symptom of dry mouth, those with Sjogren’s syndrome may experience symptoms related to swallowing function. There is potential for salivary changes to result in difficulties swallowing, which may impact other aspects of life.  What is the Purpose of this Study? This study aims to identify differences in the quantity and composition of saliva produced in those with Sjogren’s syndrome by comparing saliva samples to those of healthy individuals. In addition, we intend to explore the potential effects of changes in saliva on perceptions of swallowing. This information may help to inform future treatment methods for both swallowing dysfunction and the symptom of dry mouth. The results of this study may be used as a guide for larger studies, although there is no guarantee that they will be conducted.  A total of 40 participants will be included in this study at the University of British Columbia.  Who Can Participate in this Study? You may be able to participate in this study if you are 18 years or older, have not been diagnosed with Sjogren’s syndrome, have the ability to communicate fluently in English, and meet the requested age and sex criteria.  Who Should not Participate in this Study? You cannot participate in this study if you have a recent smoking history, prior history of radiotherapy or surgery for head and neck cancer, neurological and/or neurodegenerative disease, or currently use medications which result in reduced salivation.    85         Version: January 28, 2019  Page 3 of 6 H18-02328  What Does the Study Involve?  If you agree to participate in this study and sign this form, your saliva will be collected by trained researchers using methods described below. You will also participate in an interview regarding salivation and swallowing.  Oral mechanism examination: Prior to saliva a collection, a trained researcher will conduct an examination of your oral cavity, including both the appearance of oral structures and their function. This is estimated to require approximately 10 minutes of your time.  Assessment of oral dryness: Your oral cavity will also be assessed to determine the level of dryness. This will include assessment of the appearance of your saliva, and the appearance and adhesiveness of your oral structures. This is estimated to require approximately 5 minutes of your time.  Saliva collection: Your saliva will be collected in three different ways. You will be requested to refrain from eating, smoking, chewing gum, brushing teeth, using dental floss, and drinking anything but water for 1.5 hours prior to saliva collection (consumption of tap water allowed up to 15 minutes prior to collection). If you utilize oral moisturizers, saliva substitutes, or salivary replacement medications, you will be asked to consult with your physician and, if permitted, requested to temporarily discontinue the use of these products 48 hours prior to saliva collection. Saliva will be collected while you are sitting upright and refraining from speaking and swallowing. This is estimated to require approximately 30 minutes of your time.  Beginning with the collection of unstimulated saliva, you will be requested to expectorate all fluid within your mouth through a straw-like saliva collection aid into a tube every 30 seconds for a total duration of 2 to 5 minutes. Following a two-minute break, you will be requested to place a swab under your tongue for a total duration of 2 to 5 minutes, after which the swab will be placed into a storage tube. Stimulated saliva will be collected following another two-minute break. You will be requested to chew on a second oral swab for a total duration of 2 to 5 minutes, after which the swab will be placed into a second storage tube.   All saliva capture tubes will be immediately stored on ice in the Swallowing Innovations Lab. Following which, they will be transported in a cooler to the Weinberg Lab at UBC, and frozen until subsequent analyses are completed. The samples will be analysed to determine saliva quantity and quality using a variety of tests including assays. Saliva samples will be identified by your study participation number and not linked to your name or personal information. Samples will be stored until our data collection is completed or February 2019, whichever comes first. Following which, samples will be destroyed.  Interview: Following saliva collection, you will be asked some questions regarding your experiences with a focus on salivation, swallowing, and other impacts to the quality of your life. This is estimated to require approximately 60 minutes of your time.  The interview will be audio recorded and then transcribed. Your information will be stored using a unique study identifier and without using your name, so none of your information will be identifiable.  All data pertaining to this study will be stored in a locked facility, only accessible by study personnel.  Quality of life questionnaire: You will also be asked to complete a 44-item questionnaire regarding your feelings toward swallowing, your swallowing symptoms, and related impacts to your quality of life. This is estimated to require approximately 10 minutes of your time. This questionnaire will be   86         Version: January 28, 2019  Page 4 of 6 H18-02328  provided to you upon enrolling in the study and can be completed in advance or on the day of your study participation at UBC.  What are the Possible Harms and Discomforts? You may experience some mild physical discomfort during the collection of saliva as related to the symptom of dry mouth, limitations to oral intake prior to saliva collection, and the need to refrain from speaking and swallowing for the duration of saliva collection. You may experience some emotional discomfort during the interview while expressing your perceptions of the impacts of any oral symptoms on other aspects of your life. You will have the option to take breaks and you will not have to answer any questions that you are uncomfortable with.  What are the potential Benefits of Participating? There may be no direct benefit to you from taking part in this study. However, the information gathered from the assessments may help researchers and healthcare professionals to better understand and manage salivary and swallowing changes associated with Sjogren’s syndrome. We hope that the information learned from this study can be used in the future to benefit other people with Sjogren’s syndrome.    What are the Alternatives to Participating in this Study?  If you decide not to give consent for participation in this study your decision will in no way affect the current management from your healthcare providers or the quality of care that is provided.  What if New Information Becomes Available that May Affect My Decision to Participate? You will be advised of any new information that becomes available that may affect your willingness to remain in this study.  What Happens if I Decide to Withdraw my Consent to Participate?  You may withdraw from this study at any time without giving reasons. If you choose to enter the study and then decide to withdraw at a later time, you have the right to request the withdrawal of your information collected during the study. This request will be respected to the extent possible. Please note however that there may be exceptions where the data will not be able to be withdrawn, for example, where the data is no longer identifiable (meaning it cannot be linked in any way back to your identity) or where the data has been merged with other data. If you would like to request the withdrawal of your data, please let your study doctor know. If your participation in this study includes enrolling in any optional studies, you will be asked whether you wish to withdraw from these as well.  Will my Taking Part in this Study be Kept Confidential?  Your confidentiality will be respected. However, research records and health or other source records identifying you may be inspected in the presence of the Investigator or his designate by representatives of the University of British Columbia Clinical Research Ethics Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law. Deidentified data gathered during this study will be used in research publications in medical journals and/or scientific presentations. Study data will be kept for 25 years, as per national and institutional recommendations, and then destroyed.  You will be assigned a unique study number as a participant in this study. This number will not include any personal information that could identify you (e.g., it will not include your Personal Health Number, SIN, or your initials, etc.). Only this number will be used on any research-related information collected   87         Version: January 28, 2019  Page 5 of 6 H18-02328  during the course of this study, so that your identity (i.e. your name or any other information that could identify you) as a participant in this study will be kept confidential. Information that contains your identity will remain only with the Principal Investigator and/or designate. The list that matches your name to the unique study number that is used on your research-related information will not be removed or released without your consent unless required by law.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information. Further details about these laws are available on request to your study doctor.   What happens if something goes wrong? By signing this form, you do not give up any of your legal rights and you do not release the study doctor, participating institutions, or anyone else from their legal and professional duties. If you become ill or physically injured as a result of participation in this study, medical treatment will be provided at no additional cost to you. The costs of your medical treatment will be paid by your provincial medical plan.  What Will the Study Cost Me?  You will not incur any personal expenses as a result of partaking in this study. Following your participation in the study, you will be compensated $20.  Who do I Contact if I Have Questions About the Study During My Participation? If you have any questions or desire further information about this study before or during participation, or if you experience any adverse effects, you can contact Dr. Stacey Skoretz at 604-822-5482.  Who Do I contact if I Have Concerns About My Rights as a Study Participant? If you have any concerns or complaints about your rights as a research participant and/or your experiences while participating in this study, contact the Research Participant Complaint Line in the University of British Columbia Office of Research Ethics by e-mail at  RSIL@ors.ubc.ca or by phone at 604-822-8598 (Toll Free: 1-877-822-8598). Please reference the study number [H18-02328] when calling so the Complaint Line staff can better assist you.      88      Version: January 28, 2019  Page 6 of 6 H18-02328  CONSENT TO PARTICIPATE  Saliva Production and Composition and the Perception of Swallowing Function in Sjogren’s Syndrome  My signature on this consent form means: ▪ I have read and understood the participant information and consent form.  ▪ I have had the opportunity to ask questions and have had satisfactory responses to my questions.  ▪ I understand that participation in this study is voluntary ▪ I understand that I am completely free at any time to refuse participation or to withdraw the participant from this study at any time, and that this will not change the quality of care that he or she receive. ▪ I authorize access to my health record as described in this consent form.  ▪ I understand that I am not waiving any of my legal rights as a result of signing this consent form.  I will receive a signed copy of this consent form for my own records.  I consent to participate in this study.    _________________________________________________________________________________  Printed name of participant     _________________________________________________________________________________  Participant’s        Date Signature     _________________________________________________________________________________  Signature of Person   Printed name       Role   Date Obtaining Consent      89 Appendix E: Table of Study Operational Requirements Study component Duration Personnel Equipment Costs Per Participant Cumulative Study conduct   Recruitment 0.50 h 6 h VHL n/a $120.00*† Consenting 0.25 h 3 h VHL n/a $60.00*† Data collection 1 h 12 h VHL n/a $240.00*† Preliminary sample analyses 1 h 12 h VHL n/a  $240.00*† Immunoassays n/a 21 h W-Lab staff n/a $1,470.00*§ Salivary bioscience training n/a 24 h VHL, SAS,  IISBR staff n/a $5,000.00 W-Lab training n/a 3 h VHL, W-Lab staff n/a $210.00*§ Subtotal: 2.75 h 81 h n/a n/a $7,340.00a   Laboratory usage       Si-Lab  Consent & data collection  1.75 h  21 h  VHL  Laboratory space  $525.00*† W-Lab Preliminary sample analyses  Immunoassays 1 h   n/a 12 h   21 h VHL   W-Lab staff Laboratory space   Laboratory space $300.00*§   $525.00*§        Subtotal: 2.75 h 54 h n/a n/a $1,350.00b        Note. VHL = graduate student researcher, SAS = thesis supervisor, IISBR = Institute for Interdisciplinary Salivary Bioscience Research, Si-Lab = Swallowing Innovations Lab, W-Lab = Weinberg Lab, EIA = enzyme immunoassay, ELISA = enzyme-linked immunosorbent assay, CRP = C-reactive protein, MUC5B/7 = mucins 5B and 7, BCA = bicinchoninic acid.  aStudy conduct subsidized by $2,340.00 in-kind contributions. bLaboratory usage subsidized fully in-kind.  *In-kind contribution. †Si-Lab. ‡SASS. §W-Lab.    90 Study component Duration Personnel Equipment Costs Per Participant Cumulative Equipment   Recruitment, consenting, & data collection 1.75 h 21 h VHL Computer Documentation (printing) Digital recording device Oral cavity assessment supplies Saliva collection supplies Participant compensation  $120.00*† $20.00*† $75.00*‡ $50.00 $600.00 $140.00  Preliminary sample analyses 1 h 12 h VHL Centrifuge, laboratory balance pH test strips Aliquot tubes, micropipette tips  $240.00*§ $12.00 $100.00 Immunoassays n/a 21 h W-Lab staff Freight (kit shipping) Kinetic Reaction Kit (α-amylase) Cortisol EIA Kit ELISA Kit (CRP) MUC5B ELISA Kit MUC7 ELISA Kit BCA Protein Assay Kit $250.00 $630.00 $630.00 $1,058.00 $967.50 $967.50 $350.00       Subtotal: 2.75 h 54 h n/a n/a $6,210.00c             Grand Total 8.25 h 189 h n/a n/a $14,900.00d        Note. VHL = graduate student researcher, SAS = thesis supervisor, IISBR = Institute for Interdisciplinary Salivary Bioscience Research, Si-Lab = Swallowing Innovations Lab, W-Lab = Weinberg Lab, EIA = enzyme immunoassay, ELISA = enzyme-linked immunosorbent assay, CRP = C-reactive protein, MUC5B/7 = mucins 5B and 7, BCA = bicinchoninic acid.  aStudy conduct subsidized by $2,340.00 in-kind contributions. bLaboratory usage subsidized fully in-kind. cEquipment subsidized by $455.00 in-kind contributions. dGrand total subsidized by $4,145.00 in-kind contributions. *In-kind contribution. †Si-Lab. ‡SASS. §W-Lab.   

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