<p><strong>Introduction</strong></p> <p>Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA₂b and RyRs play a role in ASM excitation-contraction coupling.</p> <p><strong>Aim</strong></p> <p>We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.</p> <p><strong>Methods</strong></p> <p>Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10^-4M), src kinase family inhibitors PP1 (10^-5M) and PP2 (10^-5M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.</p> <p><strong> </strong></p> <p><strong>Results</strong></p> <p>Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA₂b, RyR1 and RyR2 were unchanged by the house dust mite treatment.</p> <p><strong>Conclusion</strong></p> <p>These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca²⁺-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.</p> / Master of Science (MSc)

Introduktion: Ambulanssjukvården ställer höga krav på ambulanssjuksköterskans kunskap att möta olika patienter i olika tillstånd. Ett barn med akut hindrad luftväg är en patient som är sällsynt prehospitalt, vilket många ambulanssjuksköterskor anser är det mest utmanande de kan träffa på i arbetet. En av de viktigaste kompetenserna ambulanssjuksköterskan skall ha är att kunna upprätthålla en fri luftväg, vilket leder till en stor utmaning hos ambulanssjuksköterskan om det gäller barn med akut hindrad luftväg. Det är ringa utforskat i ämnet, både i Sverige och i världen, hur upplevelserna är kring den situationen. Syftet: Att belysa ambulanssjuksköterskans upplevelser av att omhänderta barn med akut hindrad luftväg. Metod: En kvalitativ intervjustudie genomfördes med tio ambulanssjuksköterskor där insamlad data analyserades enligt Graneheim och Lundmans kvalitativa innehållsanalys. Resultatet: Sju kategorier framkom, där den mentala förberedelsen beror på kvaliteten och kvantiteten av information, omhändertagandet upplevs som enkelt, problematiskt och kreativt utmanande. Känslorna har stor spännvidd, där erfarenhet och utbildning ökar tryggheten samtidigt som ambulanssjuksköterskan känner av ett resursbehov när hen stöter på barn med akut hindrad luftväg. Konklusion: Barn med akut hindrade luftvägar utmanar ambulanssjuksköterskan maximalt kunskapsmässigt och känslomässigt, samt att ambulanssjuksköterskan önskar att få mer fortbildning inom området. / Introduction: The prehospital care in Sweden has high demands on the ambulance nurses’ qualifications to meet patients in different ages and conditions. A child with an obstructed airway is a rare type of patient in the prehospital environment, a patient which many ambulance nurses’ thinks is the most challenging they could care for. One of the essential qualifications an ambulance nurse should have is airway management, which leads to a big challenge for the ambulance nurse when it comes to children with an obstructed airway. There have been rather few studies regarding the experiences concerning these types of situations. Purpose: The purpose of the study was to enlighten how ambulance nurses’ experiences of caring for children with an obstructed airway. Method: A qualitative interview study was made with ten ambulance nurses, and the data was analyzed using Graneheim and Lundmans content analysis method. Results: The result led to seven categories. The mental preparation depends on the quality and quantity of received information. Airway management is perceived as simple, problematic and a creative challenge. The span of emotions are huge, where years of experience and education leads to feeling secure, and the ambulance nurse feel the need of human resources when working with a child with an obstructed airway. Conclusion: The conclusion is that a child with an obstructed airway challenges the ambulance nurse knowledge and emotionally to the fullest extent and there is a need for more education concerning airway management.

<p>The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.</p><p>Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.</p><p>S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. </p><p>Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.</p><p>Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.</p>

Chronic exposure to arsenic-contaminated drinking water is correlated with obstructive lung disease (i.e. chronic obstructive pulmonary disease (COPD), bronchiectasis), reduced lung function and other respiratory effects (e.g. chronic cough, chest sounds). Researchers have associated arsenic exposure with reduced airway immunity. The airway epithelial innate immune system protects underlying tissue from inhaled particulates and pathogens through a variety of mechanisms. Such defects in innate immunity are associated with chronic bacterial infections and development of obstructive airway diseases, including COPD and bronchiectasis. We hypothesize that arsenic exposure may lead to recurrent lung infection and eventual obstructive lung disease by compromising mechanisms essential in airway innate immunity. In the work presented herein we evaluated the effects of arsenic on airway epithelial barrier properties, wound repair capacity, and signaling pathways essential in innate immunity. We previously published that acute (24 hr) arsenic (0.4-3.9 μM as Naarsenite) slowed wound repair in a human bronchial epithelial cell line (16HBE14o-). In the first study we hypothesized arsenic may be affecting wound repair by altering Ca²⁺ signaling that is important in multiple aspects of wound repair, including cell migration. We found wound-induced Ca²⁺ signaling was largely mediated by paracrine ATP in 16HBE14o- cells, and acute (24 hr) arsenic (0.8, 3.9 μM) exposure reduced ATPmediated Ca²⁺ signaling. We identified functional reductions in the ATP receptors P2Y₂ and P2X₄ following arsenic exposure. Both of these receptors are essential in airway innate immunity (e.g. mucociliary clearance). In the second study we found similar reductions in wound repair capacity and ATP-mediated Ca²⁺ signaling in 16HBE14o cells using a chronic (4-5 week) low-dose (0.13, 0.33 μM) arsenic exposure representative of U.S. drinking water standards. Further, wound-induced Ca²⁺ signaling was reduced in primary cultured tracheal cells derived from mice fed arsenic-free or arsenic-supplemented (50 ppb; 1μM=75 ppb) water for four weeks prior to experimentation. In the last study we demonstrated that the structure and function of the airway epithelial barrier was altered by a five-day exposure of arsenic (0.8, 3.9 μM). We conclude that arsenic at environmentally relevant levels compromises key functions in airway epithelial innate immunity that may underlie development of lung disease.

Mast cell progenitors originate from the bone marrow and migrate to the lungs via the blood. During maturation, these cells acquire granules that contain a potent array of bronchoconstrictive mediators. The number of pulmonary mast cells is augmented in asthmatic patients and in mice with allergic airway inflammation, possibly contributing to airway hyperreactivity. An increase in mast cells is likely due to an increased recruitment of committed mast cell progenitors from the blood. However, until now a committed mast cell progenitor population has not been found in adult peripheral blood. We isolated Lin- c-kithi ST2+ integrin β7hi CD16/32hi progenitors from murine blood and showed that these cells were committed to the mast cell lineage. Based on the expression of FcεRI, these cells were less mature in Th1-prone C57BL/6 mice than in Th2-prone BALB/c mice. Asthma is associated with elevated levels of IgE. Upon exposure to allergens, IgE immune complexes are formed. In a mouse model of allergic airway inflammation, we showed that intranasal administration of IgE immune complexes to antigen-sensitized mice resulted in an increased number of mast cell progenitors compared with antigen administration alone. The increase in mast cell progenitors was independent of the low-affinity IgE receptor CD23. Rather, signaling through the common FcRγ-chain was required to enhance the number of lung mast cell progenitors. Signaling through FcεRI was likely responsible for the increase. However a role for FcγRIV could not be excluded. CD11c+ cells, such as dendritic cells, are important for antigen sensitization. In a mouse model of allergic airway inflammation, these cells are also important for the development of airway hyperreactivity, eosinophilia and Th2 cytokine production in response to antigen challenge. We showed that CD11c+ cells are critical for the recruitment of lung mast cell progenitors and the subsequent increase in mast cells. These CD11c+ cells were needed for the upregulation of endothelial vascular cell adhesion molecule-1 (VCAM-1), which is a prerequisite for the antigen-induced recruitment of lung mast cell progenitors.

Obstructive Sleep Apnea (OSA) is becoming an increasingly prevalent health problem, affecting 4% of men and 2% of women in North America. OSA is associated with many debilitating side-effects and co-morbidities; the most common being excessive daytime sleepiness (EDS), which effects the majority of OSA sufferers. EDS is negatively associated with physical activity (PA) and exercise. As a result, EDS may decrease the levels of PA performed by OSA patients. Previous research has revealed that the OSA population engages in less physical activity than the average healthy population. Studies show that CPAP treatment positively impacts EDS, and therefore; may impact PA. The primary purpose of this study was to objectively measure OSA patients' PA levels prior to CPAP treatment and 8 weeks after treatment initiation to assess whether CPAP treatment' impacts PA levels.Actigraph GT 1 M measures PA was assessed at baseline (prior to CPAP) and 8-weeks after. initiation of CPAP treatment. At each time frame, cardiovascular., blood data, body composition, and maximal cycle ergometer exercise measures were obtained. Also, subjective questionnaires, 1 reflective of sleep apnea and 1 regarding PA, were completed by the subjects.Six male subjects with severe OSA (AHI = 41.2 ± 28.4 events/hr) started and completed the study. No significant changes occurred in PA, represented as steps/day nor mean activity counts/day, throughout the 8 weeks of CPAP treatment. Significant changes were found in diastolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, and Epworth sleepiness scale scores. No significant changes occurred in any other body composition, heart rate, systolic blood pressure, triglycerides, and blood glucose. Exercise parameters, total test time, peak Watts, and V02max trended toward an increase and maximal heart rate and blood pressure toward a decrease, but none changed significantly.In conclusion, these results demonstrated that 8 weeks of CPAP treatment was not successful in increasing PA levels of severe OSA patients. The OSA subjects were categorized as sedentary according to their steps/day. Compliance to CPAP could have been an issue with subjects' average nightly usage ranging from 1.85 – 6.6hours/night. Consequently, more research regarding OSA patients PA habits and CPAP treatments effects on PA should be investigated. / School of Physical Education, Sport, and Exercise Science

Tracheal intubation in critically ill patients is a high-risk procedure. The risk of complications increases with repeated or prolonged attempts, making expedient first attempt success the goal for airway management in these patients. Patient-related factors often make visualization of the airway and placement of the tracheal tube difficult. Physiologic derangements reduce the patient's tolerance for repeated or prolonged attempts at laryngoscopy and, as a result, hypoxaemia and haemodynamic deterioration are common complications. Operator-related factors such as experience, device selection, and pharmacologic choices affect the odds of a successful intubation on the first attempt. This review will discuss the 'difficult airway' in critically ill patients and highlight recent advances in airway management that have been shown to improve first attempt success and decrease adverse events associated with the intubation of critically ill patients.

Doctor of Philosophy / Department of Kinesiology / Craig A. Harms / The overall aim of this dissertation was to determine the impact of lifestyle (i.e. habitual and acute physical activity and diet), age, and sex on systemic and airway inflammation and oxidative stress. In study 1 (Chapter 2) we examined the impact of habitual physical activity level on the post-prandial airway inflammatory response following an acute bout of moderate intensity exercise. Results indicated that the mean exhaled nitric oxide (eNO; marker of airway inflammation) response increased for all groups at two hours post high-fat meal (HFM) (~6%) and returned to baseline by four hours post-HFM. However, there was a varying eNO response from baseline to four hours in the group that exercised in the post-prandial period compared to the group that remained sedentary. These findings suggest airway inflammation occurs after a HFM when exercise is performed in the post-prandial period, regardless of habitual physical activity level. In study 2 (Chapter 3) we investigated the post-prandial oxidative stress response to meals of varying calories and fat. Specifically, we assessed the post-prandial airway and systemic 8-isoprostane (a marker of oxidative stress) responses to meals with moderate-fat (8.5 kcal/kg of bodyweight) and high-fat content (17 kcal/kg of bodyweight) from baseline to six hours post-meal in a randomized crossover design. This study revealed that systemic 8-isoprostane increased from baseline to six hours post-meal (38.3%), but there was no difference between the moderate-fat meal (MFM) and HFM conditions. There were no changes in airway 8-isoprostane from baseline to six hours post-MFM or HFM, or between the MFM and HFM conditions. Lastly, in study 3 (Chapter 4), we were interested in examining 8-isoprostane responses in older adults, since 8-isoprostane has been reported to increase with age. Previous research also suggests that older women (OW) and older men (OM) have differences with regard to prevalence and severity of late-onset asthma. In this study, we sought to determine whether the airway 8-isoprostane response to a strenuous bout of exercise was different in OW compared to OM. A secondary aim was to determine whether post-exercise 8-isoprostane generation was correlated with decrements in lung function. Our results showed that the generation of 8-isoprostane from pre- to post-exercise increased ~74±77% in OW and decreased ~12±50% in OM. The decrease in 8-isoprostane generation was not correlated with improvements in lung function from pre- to post-exercise. These findings collectively contribute to the literature by enhancing our understanding of the impact of lifestyle factors, age and sex on modifying and potentially mitigating the risk of developing chronic diseases.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by persistent sinonasal inflammation and sinus microbiome dysbiosis. The basis of this heterogeneity is poorly understood. We sought to address the hypothesis that a limited number of compositionally distinct pathogenic bacterial microbiota exist in CRS patients and invoke discrete immune responses and clinical phenotypes in CRS patients. Results: Sinus brushings from patients with CRS (n = 59) and healthy individuals (n = 10) collected during endoscopic sinus surgery were analyzed using 16S rRNA gene sequencing, predicted metagenomics, and RNA profiling of the mucosal immune response. We show that CRS patients cluster into distinct sub-groups (DSI-III), each defined by specific pattern of bacterial co-colonization (permutational multivariate analysis of variance (PERMANOVA); p = 0.001, r(2) = 0.318). Each sub-group was typically dominated by a pathogenic family: Streptococcaceae (DSI), Pseudomonadaceae (DSII), Corynebacteriaceae [DSIII(a)], or Staphylococcaceae [DSIII(b)]. Each pathogenic microbiota was predicted to be functionally distinct (PERMANOVA; p = 0.005, r(2) = 0.217) and encode uniquely enriched gene pathways including ansamycin biosynthesis (DSI), tryptophan metabolism (DSII), two-component response [DSIII(b)], and the PPAR-gamma signaling pathway [DSIII(a)]. Each is also associated with significantly distinct host immune responses; DSI, II, and III(b) invoked a variety of pro-inflammatory, T(H)1 responses, while DSIII(a), which exhibited significantly increased incidence of nasal polyps (Fisher's exact; p = 0.034, relative risk = 2.16), primarily induced IL-5 expression (Kruskal Wallis; q = 0.045). Conclusions: A large proportion of CRS patient heterogeneity may be explained by the composition of their sinus bacterial microbiota and related host immune response-features which may inform strategies for tailored therapy in this patient population.

Rationale: Tissue Factor (TF) is a transmembrane glycoprotein that canonically functions as the initiator of the coagulation cascade. Increased levels of TF have been associated with inflammatory airway diseases. Since lipopolysaccharide (LPS) is known to elicit and inflammatory response in airway epithelium, we hypothesized that airway epithelial cells release TF when exposed to LPS. Since TF aids in local wound healing, we also hypothesized that inhibition of TF would decrease NHBE growth. The specific aim of this work was to evaluate the effects of LPS exposure on TF production and release from airway epithelia and determine the signaling pathways involved. A secondary aim was to evaluate the effects of TF inhibition on NHBE growth. Methods: Normal human bronchial epithelial cells were grown in submerged cell culture and exposed to LPS as well as several intracellular signaling pathway agonist and inhibitors. Measurements: Tissue Factor mRNA and protein were measured in culture media and cell lysate by reverse-transcriptase polymerize chain reaction and enzyme-linked immunosorbent assay, respectively. Signaling pathways were evaluated using selective agonists and inhibitors. Main results: TF protein levels increased nearly two-fold in cell media after exposure to LPS (p < 0.01). This did not occur in the presence of an MEK/ERK inhibitor (PD98059) or a SMAD inhibitor (SB431542). TF protein levels also increased nearly ten-fold in the presence of TGF-beta (p < 0.05). mRNA of TF and TGF-beta was not altered by LPS or TGF-beta exposure. NHBE grown in the presence of Tissue Factor Pathway Inhibitor grew significantly slower than those grown in standard media (P < 0.05). Conclusions: NHBE release TF when exposed to LPS. This phenomenon is post-translational and may be mediated by an autocrine mechanism involving MEK/ERK signaling that increases TGF-beta which then leads to the release of TF. Our data suggest that this airway epithelium release of TF serves as a local repair function.