<p><strong>Introduction: </strong>Obesity is associated with asthma and airway hyperresponsiveness, though the mechanisms behind this relationship remain unclear. It is unlikely to be due to a direct effect of leptin on human airway smooth muscle cells (ASMC) (Nair, <em>et al.</em>, 2009). Since adipocytes are known to produce a wide array of mediators, we hypothesized that adipocytes may directly modulate human ASMC biology.</p> <p><strong>Objectives:</strong> To determine and compare the effects of intra and extrathoracic adipocyte secretions on ASMC proliferation, chemotaxis, contractility and cytokine synthesis.</p> <p><strong>Methods:</strong> Human ASMC and human adipocytes were cultured from primary samples (intrathoracic or extrathoracic). Adipocyte-conditioned media was used as a treatment in proliferation cell count assays, Transwell migrations, muscle bath experiments and to induce interleukin (IL)-6, tumor necrosis factor (TNF)-α and eotaxin production (as measured with a Bioplex). The effects of adipocyte-myocyte co-culture were also investigated on the proliferation, migration and cytokine synthesis of the ASMC.</p> <p><strong>Results: </strong>Adipocyte supernatants and co-culture did not significantly affect the growth of ASMC in the presence of 10% fetal calf serum. The adipocyte supernatants were not chemotactic, and did not affect the migration of ASMC towards platelet derived growth factor (PDGF). Similarly, co-culture did not have any effect on ASMC chemotaxis. Cytokine synthesis was also unchanged by adipocytes. Adipocyte supernatants did not have any effect on the contractile or relaxant responses of bovine tracheal smooth muscle strips. There was no significant difference between adipocyte depot location, with intrathoracic and extrathoracic adipocytes having a similar effect.</p> <p><strong>Conclusion:</strong> Human adipocytes do not directly modulate airway smooth muscle proliferation, migration, contractility and cytokine synthesis. These data point to some other cause for the association between obesity and asthma, though the role of other cells present in the adipose tissue of obese individuals cannot be ruled out.</p> / Master of Science (MSc)

Objectives: The naso- and oropharyngeal airways are influenced by a myriad of factors: jaw shape and position, tongue shape and position, lymphoid tissue, sleep apnea, chronic mouth breathing, and swallowing patterns. It is unknown if the relationships of these factors are recognized and routinely assessed in clinical orthodontics. This cross-sectional study sought to determine the proportion of participating orthodontists whom: 1) Are knowledgeable about airway restriction and its etiology, 2) Learned about these topics in post-graduate orthodontic education, 3) Consider airway restrictions in orthodontic treatment planning. Methods: A survey was administered through an online survey management platform, and sent to the email listings of 2,084 active American Association of Orthodontists (AAO) members. Survey questions are evidence-based and developed from findings in current literature. The questionnaire results were analyzed by coding and cleaning data through SAS 9.3 software. Univariate and bivariate analyses were performed to assess responses. Results: The survey received responses from 117 orthodontists. Most received their orthodontic certification from a two-year program (71.82%). The majority were knowledgeable about tongue adaptations, swallowing mechanisms, mouth breathing, and sleep apnea. Respondents were less confident about the relationship airway patency has with lymphoid tissue and with jaw position. Only half (50.51%) were taught about restricted naso- and oropharyngeal airway in post-graduate orthodontic education. A low majority, 66.32%, reported that they refer for medical consultation to the appropriate clinician before they begin treatment if a patient presents with restricted airway. Conclusions: Although the majority of respondents are knowledgeable about factors that influence airway patency, the survey identified areas in which understanding of and education in certain topics (lymphoid tissue, jaw position) may be lacking. Further emphasis should be placed on these topics to improve patient care. Orthodontics nationwide would benefit from more thorough post graduate orthodontic residency curriculum and general guidelines for clinical management of patients that present with airway obstruction. / Oral Biology

Recurrent Airway Obstruction (RAO)-affected horses experience bronchoconstriction and airway inflammation in response to inhalation of irritants including hay molds. Steaming hay reduces fungal content, but the effect on the antigenic potential has not been investigated. We tested the hypothesis that RAO-affected horses develop less severe clinical disease when fed steamed versus non-steamed hay and this reduction coincides with decreased hay fungal content. Six RAO-affected horses in clinical remission were divided in two groups and fed steamed or non-steamed hay for 10 days using a two-way cross-over design. Hay was steamed using a commercial hay-steamer. Clinical assessment was performed daily. Full assessment, including airway endoscopy, tracheal mucous scores and maximal change in pleural pressure, was performed on days 1, 5, and 10. Bronchial fluid sampling and cytology were performed on days 1 and 10. Hay core samples were collected pre- and post-steaming and cultured to determine fungal and bacterial concentrations. Statistical analysis was based on data distribution and quantity and performed using SAS®. P-value <0.05 was significant. Steaming significantly decreased the number of bacterial and fungal colony-forming-units in hay. Horses fed non-steamed hay experienced a significant increase in clinical score and a trend towards airway neutrophilia, while parameters were unchanged in horses fed steamed hay. Only horses fed non-steamed hay experienced a significant increase in tracheal mucous score. Horses fed steamed hay gained significantly more weight compared to horses fed non-steamed hay, even though the amount of hay consumed not greater on a dry matter basis. These results indicate that steaming reduces the RAO-affected horse's response to hay which coincides with a reduction in viable fungal content of hay. / Master of Science

Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions. / Master of Science

ABSTRACT Patients with respiratory failure secondary to acute asthma exacerbation (AAE) frequently present at emergency units. Some patients may develop respiratory muscle fatigue. Current guidelines for the treatment of an AAE center on pharmacological treatment and invasive mechanical ventilation. Noninvasive positive pressure ventilation (NPPV) has an established role in COPD exacerbations. The role it can play in an AAE remains unanswered although it is frequently used in the clinical setting. Aims: The present study proposed to investigate if the early use of NPPV in the forms of continuous positive airway pressure (CPAP) or bilevel positive pressure ventilation (BPPV) together with standard medical therapy in AAE can decrease time of response to therapy compared to standard medical therapy alone. We further tested the effect of BPPV against CPAP. Methods: Asthmatic patients who presented with a severe AAE (PEFR % predicted < 60 %) at the emergency unit were randomized to either standard medical therapy (ST), ST and CPAP or ST and BPPV. Thirty patients fulfilled the inclusion criteria for the study. Groups presented similar baseline characteristics. The mean age for the group was 42.1 ± 12.6 years. Mean baseline PEFR % predicted was 35.2 ± 10.7 % (ST), 30.5 ± 11.7 % (ST + CPAP) and 33.5 ±13.8 % (ST + BPPV). Results: Hourly improvement (Δ) in respiratory rate and sensation of breathlessness was significantly better in the BPPV intervention group. Improvement (Δ) from baseline to end of treatment in respiratory rate and sensation of breathlessness was significant for both CPAP and BPPV (p = 0.0463; p = 0.0132 respectively) compared to ST alone. Lung function was significantly improved in the CPAP intervention group hourly and from baseline to end of treatment (p = 0.0403 for PEFR and p = 0.0293 for PEFR % predicted) compared to ST + BPPV and ST alone. The mean shift (Δ) in PEFR from baseline to 3 hours of treatment was 67.4, 123.5 and 86.8 L/min (p = 0.0445) for ST, ST + CPAP and ST + BPPV respectively. This corresponded to a 38.1, 80.8 and 51.7 % improvement in lung function respectively. Discussion: The effect of BPPV on the reduction of respiratory rate and sensation of breathlessness could be related to the inspiratory assistance provided by BPPV. The significant improvement in lung function in the CPAP group could be related to its intrinsic effect on the airway smooth muscle and / or on the airway smooth muscle load. Conclusion: The present results suggest that adding NPPV to standard treatment for an AAE not only improves clinical signs faster but also improves lung function faster. CPAP seems to have an intrinsic effect on the airway smooth muscle so rendering it more effective in ameliorating lung function.

OBJECTIVES: This retrospective cohort study tested the effect of Rapid Maxillary Expansion (RME) on symmetrical volumetric changes in the nasal cavity. Volumetric changes in overall nasal cavity, nasopharynx, and oropharynx were also assessed as well as minimum cross-sectional width changes and molar angulation in association with RME. METHODS: CBCT scans of before and after RME treatment for 28 subjects (17 females, 11 males, average age 9.85 ± 2.42 years) were collected from a previously de-identified database. All subjects were treated for maxillary constriction using banded hyrax expanders. Mimics software was utilized to segment the nasal and pharyngeal airways and create various compartments (left and right nasal cavity, nasopharynx, and oropharynx) for volumetric analysis. Minimum cross-sectional width measurements and maxillary first molar angulation were also assessed. Paired T-test was used to quantify the changes brought about by expansion. Statistical significance was set at the 0.05 level. RESULTS: Posterior expansion as measured between right and left greater palatine foramen (GPF) averaged 2.41 mm (SD = 1.03 mm). There were statistically significant differences in overall nasal cavity (2249.6 ± 2102.5 mm3), right nasal cavity (968.8 ± 1082.7), left nasal cavity (1197.3 ± 1587.0), nasopharyngeal (1000.6 ± 917.7), and oropharyngeal (2349.2 ± 2520.8) volumes. In comparing the right to left nasal cavity, no significant changes were noted for initial volume, post-expansion volume, or pre to post-expansion changes (T2-T1). For cross-sectional analysis, the right nasal cavity (0.13 ± 0.07 mm) and left nasal cavity (0.11 ± 0.06 mm) showed significant increases in minimum crosssectional width measurements. Initial maxillary molar angulation had no significant correlation to initial nasal cavity volume on either side. CONCLUSIONS: RME has significant benefits to increasing nasal and pharyngeal airway cavity volumes in all segments of the airway. Nasal cavity expands symmetrically. Minimum cross-sectional width of the left and right nasal cavities showed highly symmetrical improvements. Initial maxillary molar angulation has no relationship to initial nasal cavity volume.

In healthy airways, mucus forms part of the innate immune response protecting the respiratory epithelium from damage by pathogens and environmental debris (Rose and Voynow, 2006). Conversely, in many respiratory diseases, mucus becomes part of the airway disease pathology. Mucus hypersecretion along with reduced clearance can cause blockage of the small airways, impairing gas exchange, promoting inflammation and becoming a culture medium for bacterial colonisation (Thornton et al., 2008). Recurrent airway obstruction (RAO) is a common yet poorly understood equine chronic respiratory disease where such altered mucus properties and clearance have been identified as major factors in the disease pathology (Davis and Rush, 2002; Gerber et al., 2000; Kaup et al., 1990; Robinson, 2001). The gel-forming mucins are largely responsible for the transport properties of mucus. The major equine airway gel-forming mucin in health is Muc5b and to a lesser extent Muc5ac; produced in specialised respiratory epithelial goblet cells and sub-mucosal glands (Rousseau et al., 2011b). Changes in mucin relative and net amounts and their macromolecular properties and interactions have been attributed to the altered physical properties of airway mucus in airways disease (Groneberg et al., 2002a; Jefcoat et al., 2001; Kirkham et al., 2002; Robinson et al., 2003; Sheehan et al., 1995).The project investigates the biochemical properties of mucins present in mucus from healthy horses and horses with RAO. This project identifies the anatomical presence of mucin-producing goblet cells and glands in fixed tissues from the respiratory tracts of healthy horses and subsequently examines mucin-production sites in respiratory tracts from horses with RAO. Finally the project investigates a methodology for the study of mucin production in airway cells harvested from live horses suffering from RAO.Our investigations confirmed that horses with RAO have more endotracheal mucus than healthy controls, and that Muc5b is the predominant mucin with Muc5ac also present in RAO horse mucus, both during symptomatic disease and when horses are asymptomatic. Mucins are produced in epithelial goblet cells and sub-mucosal glands dispersed throughout the length and circumference of the equine trachea and bronchi. Goblet cell hyperplasia occurs in symptomatic exposed RAO horse airways, although goblet cells are smaller than in asymptomatic RAO horse airways. Exposure to a dusty stable environment is associated with more goblet cells per length of bronchial compared to tracheal epithelium in all horses. RAO horses have larger sub-mucosal glands containing more mucin than control horses. Primary epithelial cell cultures grown at an air liquid interface are an alternative approach to study equine airway mucus, although the use of this culture system is in its early stages. We have developed novel ways to harvest equine airway epithelial cells (tracheal brushing) and shown it is possible to freeze cells collected via tracheal epithelial brushing in 20 % FBS and then culture to ALI at a later date.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.

Fatty acid-binding proteins are small intracellular proteins with poorly defined functions in intracellular fatty acid transport. The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. Using Affymetrix microarrays, we found that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by airway epithelial cells. aP2 expression was markedly increased following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13, and downregulated by the Th1 cytokine IFN-gamma. Regulation of aP2 mRNA expression by Th2 cytokines was dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2 deficient mice in a model of allergic airway inflammation, and found that infiltration of leukocytes, especially eosinophils, into the airways was highly aP2 dependent. T cell priming and peritoneal allergy was unaffected by aP2 deficiency suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-haematopoietic cells, most likely airway epithelial cells, as the site of aP2 action in allergic airway inflammation. Expression of the pro-inflammatory cytokines MCP-1 and IL-6 was impaired in cytokine activated aP2 deficient airway epithelial cells, while levels of the anti-inflammatory arachidonic acid metabolite 15-HETE was increased, providing a mechanism for the reduced airway inflammation in aP2 deficient mice. In addition to the immune functions of aP2, we found that the related fatty acid-binding protein mal1 was also upregulated by IL-4/IL-13 in airway epithelial cells, and mal1 deficient mice were protected against airway eosinophilia. Significantly, in comparison to single aP2 deficiency, mice with combined aP2-mal1 deficiency had augmented protection against airway inflammation, and bone marrow chimera experiments demonstrated that aP2-mal1 deficiency affected both non-haematopoeitic and haematopoeitic cells. In T cell priming experiments, aP2-mal1 deficiency resulted in defective cytokine profiles in antigen recall responses, suggesting compromised sensitisation to antigen as one mechanism for aP2-mal1 action in airway inflammation. Together, our data therefore demonstrates the crucial roles of fatty acid-binding proteins in airway epithelium, T cell priming and airway inflammation, and provides a new link between fatty acid signalling and allergy.

Human epithelium-specific ETS transcription factor-1 (ESE-1), which is also known as E74-like factor-3 (Elf3) in mice, is strongly expressed in lung during fetal development and in certain lung cancers. The primary goal of the work presented in this thesis was to investigate whether ESE-1 is involved in regeneration of the injured lung epithelium by administering naphthalene to both wild-type (Elf3 +/+) and Elf3-deficient (Elf3 -/-) mice. However, optimal conditions for proper utilization of the naphthalene-induced lung injury model must first be established. Therefore, dose-response studies were initially conducted by administering three different doses of naphthalene to both male and female mice, as described in chapter 2. Although it is shown that the extent of naphthalene-induced Clara cell injury is dose-dependent in both male and female mice, female mice are more sensitive to naphthalene-induced injury than male mice independent of the dose. Furthermore, it is also demonstrated that these gender-dependent differences in naphthalene injury can subsequently influence downstream lung repair kinetics. In light of these findings, lung regeneration was examined in both sexes of both Elf3 +/+ and Elf3 -/- mice. As reported in chapter 3, the kinetics of bronchiolar epithelial cell proliferation and differentiation is delayed considerably in Elf3 -/- mice following naphthalene injury. Moreover, expression of transforming growth factor-beta type II receptor, which is a well-known transcriptional target gene of ESE-1 and is involved in the induction of epithelial cell differentiation, is significantly lower in the bronchiolar airway epithelium of Elf3 -/- mice as compared to Elf3 +/+ mice under steady-state conditions and during repair of naphthalene-induced damage. Collectively, these findings occur to a similar extent in both sexes of both Elf3 +/+ and Elf3 -/- mice, and suggest that ESE-1 plays an important role in regulating the kinetics of airway epithelial regeneration after acute lung injury.