Cystic fibrosis (CF) is a lethal genetic disease that affects 30,000 people in the United States alone. While the disease affects organs throughout the body, it is the lung disease that is the primary cause of morbidity and mortality for people with the disease. CF lung disease is characterized by thick and sticky mucus that obstructs the airways, acute and chronic bacterial infections, and chronic inflammation and remodeling. Thanks to the creation of the CF pig, it is now possible to study the manifestations of CF lung disease at birth. The CF pig develops spontaneous lung disease, similar to that found in humans with CF, making it the ideal model for our studies. One of the critical findings that revealed in studies of the CF pig is that airway surface liquid (ASL) bactericidal activity is impaired in CF at birth, and this activity is pH dependent. Because infants and children with CF tend to suffer greater morbidity from respiratory viruses than non-CF infants and children, we sought to determine if ASL has antiviral activity and if that activity is reduced in newborn CF pigs. We found that pre-incubating either tracheal or nasal ASL from wild-type pigs reduced the infectivity of various recombinant viruses expressing an eGFP or GFP reporter gene. Those viruses include Sendai virus (SeV-eGFP), respiratory syncytial virus (RSV-GFP), the PR8 strain of influenza virus A (PR8-eGFP), and adenovirus (Ad-eGFP), indicating ASL has broad-spectrum antiviral activity. Nasal secretions from newborn CF pigs had strikingly reduced antiviral activity against SeV-eGFP and Ad-eGFP compared to nasal secretions from WT littermates. Unlike what was observed for ASL antibacterial activity, nasal secretion antiviral activity was not affected by pH, nor was it affected by bicarbonate concentration, one of the molecules that drives pH in the airways. However, when we mixed CF and WT nasal secretions at different ratios, we found the antiviral activity to follow a linear trend, with antiviral activity increasing as the percentage of WT nasal secretions increased. This suggests that one or more components of nasal secretions are found less abundantly in CF nasal secretions compared to WT nasal secretions, leading to reduced antiviral activity in CF. The CF pig has facilitated a much greater understanding of the early stages of CF lung disease. This model will allow us to determine what antiviral components are lacking in the CF airways and why they are reduced in CF.

Approximately 7.5 million people suffer from voice disorders in the United States. Previous studies indicate that the quality of the fluid layer that coats the vocal folds appears to be different for people with voice disorders than for people whose voice is considered normal. These studies suggest that the composition and/or physical properties of the fluid layer may contribute to voice disorders. Despite these findings, little research has been undertaken to investigate the role of the fluid layer on voice, and in almost all cases, the fluid layer is considered to be insignificant. The purpose of this research was to investigate the role of the fluid layer and the potential it may have to influence voice production; particularly, to identify some aspects of the fluid layer that have the potential to contribute to voice disorders. In order to investigate the potential significance of the effects of a fluid layer on vocal fold operation, an existing lumped model was modified to incorporate the Newtonian squeeze-flow equation as a fluid model during the colliding portion of the oscillatory cycle. Results indicated that thicker films produced more significant deviations from the case with no fluid layer. Experimental testing was performed to validate existing analytical equations for squeezing flow of Newtonian and non-Newtonian fluids confined between parallel axisymmetric plates. Based on available published data on the rheological properties of the fluid layer found on the surface of the vocal folds, several fluids with a range of fluid properties were selected. Reasonable agreement was found for much of data collected for the Newtonian fluid cases within measurement tolerances. For the non-Newtonian cases, the constitutive equation was found to be in poor agreement with the measured physical characteristics of the selected non-Newtonian fluids. A summary of the collected experimental data is provided so that it can be used in for validation and comparison in future research. A preliminary computational model based on the classical two-mass vocal fold model was implemented which incorporated squeezing effects of a thin Newtonian film of fluid on the surface of the vocal folds. Results indicated that the fluid layer may not be insignificant, although further tests and modeling are required. Finally, different fluids were applied to a physical model of the vocal folds and measurements were taken to determine the effects of the application of fluid. The results showed significant changes in the vocal fold model response that indicated the fluid layer affects vocal fold operation in important ways. Some of the changes in response could not be attributed solely to the fluid layer. Suggestions regarding future work with physical model testing are given which may help clarify the effects of a fluid layer on vocal fold flow-induced vibration.

The airway surface contains a number of important defense mechanisms to protect against infection. Antimicrobials found in the thin layer of fluid lining the airways, the airway surface liquid (ASL), rapidly kill bacteria. Another defense mechanism, mucociliary transport, propels foreign particles and mucus out of the airways. These and potentially other host defense properties show a dependence on the pH of the ASL. An acidic ASL pH reduces bacterial killing by cationic antimicrobial peptides, and increases ASL viscosity, potentially effecting mucociliary transport. Consequently, an acidic ASL pH can impair airway host defense. An example of a disease where an acidic ASL pH impairs airway host defense, is Cystic Fibrosis (CF). The major cause of morbidity and mortality in CF is airway infections. Humans with CF and the CF pig model develop airway infections. But curiously CF mice are spared. Compared to non-CF, people with CF and CF pigs show an abnormally acidic ASL pH. However, the ASL pH in CF mice is not different to that of non-CF. Thus, we hypothesized that CF mice do not show defects in airway host defense because their ASL pH is not acidic compared to non-CF. As pH is a balance between acid and base secretion, we first determined which HCO3- and H+ secreting proteins contribute to ASL pH and the differences between humans, pigs, and mice. CF is caused by defects in an anion channel, CFTR. We found that in all three species, CFTR secreted HCO3- into the ASL, which was absent when CFTR was defective. To determine how much CFTR is required to rescue ASL pH and host defense properties, we mixed CF and non-CF airway epithelia from newborn pigs in different ratios. HCO3- secretion, ASL pH, and host defense properties showed a direct relationship to CFTR, suggesting that CFTR was the rate-limiting step. As CFTR was limiting, we found that supernormal CFTR expression in a small number of cells could dramatically increase ASL pH, suggesting viral-mediated gene therapy approaches may have benefit for CF. We found that Ca2+ activated Cl- channels also played some role in ASL pH in CF pigs and CF mice. However, as CF pigs develop airway infection, while CF mice do not, this suggested that other factors might be important for differences in ASL pH and consequently the development of disease. To further investigate the determinants of ASL pH, we examined H+ secretion. Humans and pigs showed 6 times more H+ secretion compared to mice. This acidification occurred through the non-gastric H+/K+ ATPase (ATP12A). ATP12A was also much more highly expressed in human and pig airways compared to mice. Blocking ATP12A in human and pig airways increased ASL pH and consequently improved host defense properties such as bacterial killing and ASL viscosity. Conversely, expressing ATP12A in CF mouse airways acidified ASL, impaired defenses, and increased airway bacteria. These findings suggest that ASL pH is a balance between HCO3- and H+ secretion. In humans and pigs lacking CFTR, unchecked H+ secretion by the non-gastric H+/K+ ATPase (ATP12A) acidifies the ASL, which impairs airway host defenses. However, as mouse airways expressed little ATP12A and secrete minimal H+, loss of CFTR does not lead to ASL acidification, protecting CF mice. These findings not only help explain why CF mice are protected from infection, but also nominate ATP12A as a therapeutic target for improving ASL pH and host defense in humans with CF.

Cystic fibrosis (CF) is an inherited disease with symptoms mainly in the respiratory tract. The airway epithelium is covered with a thin layer of fluid, the airway surface liquid (ASL). The volume and composition of ASL are important in the pathogenesis of cystic fibrosis. The composition of ASL was determined. Firstly, pig airways were analyzed by X-ray microanalysis in the frozen-hydrated state. Secondly, small Sephadex beads were left to absorb the ASL in situ and were analyzed by X-ray microanalysis. The Na and Cl concentrations in the ASL of the pig were close to those of these ions in serum. Rat tracheal ASL was hypotonic. However, rat nasal fluid was hypertonic with an extremely high concentration of K. The composition of the ASL could be influenced by pharmacological stimulation. The development of transgenic mouse models for CF may help to develop therapies for the disease. The composition of mouse ASL was investigated using different collection techniques. (1) beads mounted on filter paper, (2) beads randomly spread over the airway epithelium, and (3) beads spread over the epithelium with a syringe. No significant difference could be detected between these techniques, and mouse ASL was hypotonic. Calibration curves had to be made for each element of interest. Nasal fluid from healthy human volunteers was collected with: (1) a pipette, (2) filter paper, (3) cotton wool, or (4) Sephadex beads. Collection on filter paper and equilibration with Sephadex beads gave reliable results. The Na and Cl concentrations in nasal fluid of control subjects were about the same as in serum, but the K concentration was higher. Rhinitis or primary ciliary dyskinesia patients and CF heterozygotes had abnormally high concentrations of Na and Cl in their nasal fluid (probably due to inflammation of the nasal epithelium), and CF homozygotes had even higher concentrations of Na and Cl.

The vocal folds are a complex self-oscillating biological system. In the current research, an equation was developed to model viscous adhesion forces that occur when the collision of the vocal folds results in the formation of a liquid bridge. The adhesion equation was validated using experimental data, and simplified to a one-dimensional approximation with an included correction factor that adjusted the predicted pressure in situations where the one-dimensional approximation was invalid. A non-oscillating vocal fold model with a modeled liquid bridge was used to study stress resulting from viscous adhesion. The vertical normal stress magnitude ranged from about 80 to 1700 Pa. This was shown to be of the same order of magnitude as the stress due to collision of the vocal folds. Also the stress resulted in large normal strains that occurred at small distances below the surface of the vocal folds consistent with lesion development. Therefore, it was determined that the viscous adhesion may be a contributor to damage of the vocal folds that leads to the development of benign lesions, such as vocal nodules. This conclusion was further validated by adding the adhesion equation in a self-oscillating vocal fold model. The influence of adhesion on the dynamics of the model was significant. The frequency of vibration was reduced by nearly 2.5% for the case of adhesion with a mucus viscosity of 0.01 Pa-s. Also adhesion induced positive tensile stress that resulted in normal strain distributions similar to those seen in the non-oscillating cases. These results also indicated that liquid mediated viscous adhesion may be a contributor to the development of benign lesions (nodules). However, further research is needed to validate these conclusions.

Cystic fibrosis (CF) is one of the most common fatal inherited diseases, most prevalent among Caucasians. CF is caused by a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), which helps to create sweat, digestive juices, and airway surface liquid (ASL). The airways are covered with a thin layer of fluid, the airway surface liquid, in which the cilia bathe. Defective or absent CFTR leads to a defective water and ion transport in the epithelial cells, which results in viscous mucus, defective ciliary clearance, bacterial inflammation and tissue damage. The volume and composition of ASL are important in the pathogenesis of cystic fibrosis and it is therefore relevant to determine its composition. However, there are a number of difficulties in determining the ionic composition of the ASL due to its small volume. Literature data vary from very hypotonic to markedly hypertonic. These controversial data inspired the development of a simple method for determining the elemental composition of the ASL in different animal species and humans. Two techniques were developed to determine the composition of ASL, from which indirect information on chloride transport in airway epithelium can be obtained. In the first method, tissue is removed from the animals under anesthesia, frozen and analyzed in the frozen-hydrated state. In the second method, the ASL is collected with small dextran (Sephadex) beads; the dried beads are then analyzed by X-ray microanalysis. The Sephadex-bead method appears more accurate compared to the frozen-hydrated samples. Both methods were applied to collect tracheal and/or nasal fluid in pigs, normal and transgenic cystic fibrosis mice, the fluid covering the apical surface of normal bronchial cells (16HBE14o-) and a cystic fibrosis human bronchial cell line (CFBE41o-), and finally nasal fluid in healthy and diseased subjects. The ionic composition of the ASL was isotonic both in pigs and healthy human subjects. CF patients had much higher levels of Na and Cl ions than healthy subjects. The ASL under control conditions was hypotonic in mice and cell cultures, whereas the concentrations of Na and Cl ions in the species with the ΔF508 mutation or absent CFTR were significantly higher than in the corresponding controls. It was also demonstrated that the ionic composition of the ASL can be influenced by pharmacological treatment. The study confirms earlier findings that CFTR also is involved in bicarbonate transport. Mist tent therapy has been tested in the study of a treatment for CF patients, in order to hydrate the viscous mucus. But the effect of mist tent therapy on ion concentrations in the ASL appeared to be short-lived, although no patients became chronically colonized with pseudomonads while on nocturnal mist tent therapy.

La mucoviscidose est la maladie génétique autosomique récessive létale la plus fréquente dans la population caucasienne. Le gène muté code pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). CFTR est un canal anionique localisé dans la membrane apicale des épithélia. Il permet notamment le transport d’ions chlorure (Cl-) et bicarbonates (HCO3-). La délétion de la phénylalanine en position 508 (F508del) de CFTR est la mutation la plus fréquente et entraîne un défaut d’adressage de CFTR à la membrane plasmique. L’atteinte respiratoire réalise une bronchopathie obstructive surinfectée qui détermine le pronostic vital de la maladie. Deux mécanismes délétères présents dès la période néonatale vont notamment contribuer à la destruction du tissu pulmonaire : une inflammation exacerbée et auto-entretenue, et une colonisation bactérienne chronique du mucus notamment à Pseudomonas aerugonisa et Staphylococcus aureus. Les mécanismes à l’origine de l'initiation de ce double processus demeurent inconnus. De plus en plus d'arguments plaident pour l'implication des ions HCO3-, donc du pH, dans l'initiation de l'atteinte pulmonaire de la mucoviscidose. En effet, le défaut de CFTR entraîne une diminution de leur sécrétion dans le liquide de surface bronchique (ou Airway Surface Liquid : ASL), ce qui conduit à un pH local anormalement bas. Les ions HCO3- jouent un rôle important dans la physiopathologie bronchique pulmonaire. En effet, ils régulent la rhéologie du mucus et sont impliqués dans l’activité des peptides antimicrobiens, principaux facteurs de la bactéricidie locale et dont l'activité est pH dépendante. Cela a été démontré dans un modèle porcin de mucoviscidose. Ainsi une altération du transport de HCO3- pourrait contribuer à l'hyperviscosité des sécrétions muqueuses, initier la colonisation bactérienne en diminuant le pouvoir bactéricide du liquide de surface respiratoire. Cette hypothèse est la base de mon projet visant à étudier le rôle du transport transépithélial des ions bicarbonates dans la bactéricidie au sein de l’épithélium respiratoire dans la mucoviscidose. La mesure du pH de l’ASL a nécessité la conception d’une enceinte à atmosphère contrôlée. Cet outil a permis la mise en évidence d’une acidité de l’ASL F508del en comparaison du Wild Type (WT), due à un défaut de sécrétion des ions HCO3- dans l’ASL, lui-même induit par une inhibition fonctionnelle du transporteur Cl-/HCO3- SLC26A4 ou pendrine majoritairement et du canal CFTR minoritairement. L’évaluation de la bactéricidie de l’ASL après infection au Staphylococcus aureus révèle une déficience de bactéricidie chez les cellules épithéliales F508del, reliée à l’activité anormale de la pendrine. L’investigation des capacités d’adhésion et d’invasion du Staphylococcus aureus dans nos modèles montre que le défaut de bactéricidie épithélial observé se restreint à l’ASL et incite à poursuivre les recherches sur le rôle du pH de l’ASL dans l’activité de ses peptides antimicrobiens, principale ligne de défense de l’immunité innée respiratoire. Notre travail de restauration du pH de l’ASL avant infection de l’épithélium respiratoire démontre d’ores et déjà l’impact significatif du pH de l’ASL dans la modulation de sa bactéricidie, où la restauration du pH est corrélée à une amélioration de la bactéricidie. Ce travail met en lumière le rôle crucial du pH de l’ASL dans l’appréhension de la physiopathologie de la maladie et sensibilise à de nouvelles voies thérapeutiques, basées sur une restauration du pH de l’ASL et/ou l’utilisation de peptides antimicrobiens pH-indépendant. / Cystic fibrosis (CF) is a lethal autosomal recessive disorder caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene encoding for a cAMP-activated anionic channel, secreting mainly chloride (Cl-) and bicarbonate (HCO3-) at the apical surface of the epithelia. Most patients are homozygous for the p.PHe508del mutation (F508del). The main cause of morbidity and mortality is obstructive lung disease characterized by exacerbated inflammation and bacterial infection of the airway surface liquid (ASL), a thin layer coating the luminal face of the airway epithelium. ASL bacterial colonization begins from the first hours of life with evidence of Staphylococcus aureus in airway secretions pointing to impaired local defense. However, the defect responsible for this defective bacterial clearance is not clearly understood. It was proposed to be related to decreased mucociliary clearance and abnormal inflammatory responses, but recent studies also show the contribution of ASL in the reduced antimicrobial capacity of ASL in CF airways. An abnormally low ASL pH impairs mucin hydration and solubilization, resulting in hyperviscous mucus, which impedes muco-ciliary clearance. It also reduces the activity of antimicrobial peptides by modulating their native charges and the bactericidal activity of antibiotics. This was supported by studies in newborn CF pigs, which highlighted an abnormally low ASL pH, in association with a defective short-term S. aureus antimicrobial activity. Restoring normal pH in the ASL of CF pigs improved ability to eradicate the bacteria, showing that reduced ASL pH is central to disease pathogenesis. However, there is still controversy about the value of ASL pH in humans. Very recently, a study in young CF children, based on in vivo measurements, showed similar ASL pH values in children with CF to that in children without CF. The mechanisms underlying the ASL pH homeostasis and in particular the balance between HCO3- and proton (H+) secretion are still not known. Shah et al highlighted the role of persistent H+ secretion by ATP12A concomitant to a CFTR-mediated reduced bicarbonate transport, but recent work overexpressing ATP12A in respiratory cells failed to find any pH modification in physiological conditions. Most importantly, there is no clear understanding of the initial host response, when S. aureus bacteria land on the pristine surface of a newborn airway, with a prolonged time of contact and continuous reseeding from infected mucus plugs. This is however crucial to clarify pathogenesis of this early steps to counteract pro-infectious vicious circle and define optimal therapeutic strategy in newborns. We focused on human airways and hypothesized that S. aureus clearance during the first hours of infection was impaired in human airway CF ASL because of lowered ASL pH. To test this hypothesis, we designed bacterial infection experiments within human airway epithelium to mirror the onset of initial S. aureus infection. We then studied the relationship between local bacterial clearance and ASL pH regulation in WT and F508del homozygous human bronchial epithelial cells, with special emphasis on physiologically relevant HCO3- and H+ transporters.

Cystic fibrosis (CF) is the most common genetic disease affecting Canadian newborns (1 in 3,850) and is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. This gene encodes for CFTR, a phosphorylation-dependent ion channel localized at the apical membrane. Phosphorylation of CFTR by the cyclic adenosine monophosphate (cAMP)-dependent enzyme protein kinase A activates its activity, facilitating the transport of chloride and bicarbonate ions across the epithelial membrane. CFTR contributes to ion and airway surface liquid regulation, crucial for maintaining host defenses. The inheritance of CFTR mutations leads to a variety of respiratory complications, including impaired mucociliary clearance, excessive mucus production, persistent airway infections, and heightened inflammation, ultimately causing lung damage. While there is currently no cure for CF, the development of CFTR modulators, targeting the defective CFTR protein directly, has significantly improved the quality of life for many CF patients. Despite these advancements, many patients remain unresponsive to current treatment options. It has been well-established that combination therapies outperform monotherapies, emphasizing the need for alternative or complementary therapeutic strategies for CF management. Furthermore, CFTR dysfunction extends beyond CF and has been implicated in other respiratory diseases, such as chronic obstructive pulmonary disease, which is primarily linked to tobacco smoke exposure. This Ph.D. thesis explores a complementary therapeutic approach, targeting proteins within the CFTR-containing macromolecular signaling complex to elevate intracellular cAMP levels, thereby enhancing CFTR function. We hypothesized that synergistic use of cAMP modulators, alongside CFTR modulators, will serve as an effective therapeutic strategy for CF and other respiratory diseases. Collectively, our studies highlight the potential of cAMP and CFTR modulation as a therapeutic strategy for improving the treatment of CF and other respiratory diseases, warranting further investigation, offering insights for future studies, and contributes to the ongoing pursuit of improved combination treatments. / Dissertation / Doctor of Philosophy (PhD) / Cystic fibrosis (CF) is the most common genetic condition affecting Canadian newborns, caused by inheritance of mutations in the CF transmembrane conductance regulator (CFTR) gene. These mutations result in respiratory issues, including breathlessness, excess mucus, and susceptibility to infections, causing lung damage and premature death. Despite progress in CF drug development, some patients remain unresponsive to existing drug combinations, highlighting the need for new combinations to improve the quality of life for all CF patients. CFTR function is also compromised in other respiratory diseases like chronic obstructive pulmonary disease, a lung disease that shares many characteristics with CF and is mainly caused by tobacco smoke exposure. This Ph.D. thesis explores the effectiveness of a new drug strategy targeting proteins interacting with CFTR. By investigating drugs to complement existing treatments, we aim to improve CFTR function. This research offers a promising strategy to improve treatment for CF and other respiratory diseases.