Rationale: Ventilation inhomogeneity (VI) may be an early sign of obstructive airway disease. The lung clearance index (LCI) has been suggested as a sensitive marker of VI, although it has not been well characterized in young children in health and in those with CF and asthma. Objective: To determine if LCI can detect VI in asymptomatic infants and preschool-age subjects with CF or wheeze/asthma compared to healthy controls. Methods: Sulphur hexafluoride (SF6) multiple breath washout (MBW) testing was completed in all subjects. Results: LCI was found to be dependent on age in a large healthy cohort. Accounting for age, LCI was significantly elevated in disease groups compared to healthy controls in early childhood, illustrating early presence of VI in wheezy infants and the progression of disease in CF. Furthermore, the effects of breathing pattern and the variability of MBW parameters showed positive associations with age and VI.

La fibrosis quística (fq) es la enfermedad autosómica recesiva más frecuente en la población caucasoide con una frecuencia de portadores de 1/25. Las manifestaciones clínicas más importantes son las infecciones crónicas recurrentes del pulmón conllevando al deterioro del mismo. En esta tesis nos propusimos corregir dos mutaciones en el gen cftr, responsable de la fq, en la linea celular de epitelio bronquial ib3.1 de genotipo (f508del/w1282x). Para ello, mediante citometría de flujo y microscopía confocal pusimos a punto la incorporación no viral (vectores pei, geneporter y citofectina) de oligonucleótidos modificados (quimeraplastos y oligonucleótidos fosforotioato) y de fragmentos sfhr en estas células. Los quimeraplastos son oligonucléotidos quiméricos de rna y dna y los oligonucleótidos fosforotioato contienen enlaces fosforotioato para evitar su degradación además de enlaces fosfodiéster. Ambos, son capaces de estimular los mecanismos de reparación intrínsecos de las células para corregir a nivel de dna mutaciones puntuales. Los fragmentos sfhr (small fragment homologous replacement) son fragmentos de pcr de longitud variable con la secuencia salvaje del gen que se intercambian con las secuencias genómicas diana mutadas por recombinación homóloga para revertir mutaciones puntuales (cambios de 1 nucleótido, pequeñas inserciones o deleciones). Para estimar el porcentaje de corrección génica adaptamos la técnica diagnóstica de pcr-ola a nuestro modelo para utilizarla como metodología de cuantificación. Además, hemos confirmado que las células ib3.1 tienen los mecanismos de reparación de mutaciones puntuales activos tanto por rt-pcr como por un ensayo in vitro en e.coli con el plásmido pksm4021 que contiene el gen de resistencia a ampicilina y el gen de resistencia a kanamicina inactivado por una mutación puntual. Los resultados que se publicaron en los siguientes artículos nos han permitido extraer las siguientes conclusiones: las células Ib3.1 de epitelio bronquial de fq son competentes en cuanto al sistema de reparación mmr. La incorporación celular de quimeraplastos, oligonucleótidos monocadena y fragmentos sfhr es muy eficiente en las células ib3.1. El lípido catiónico citofectina, el policatión pei y la electroporación, aunque no el lípido catiónico geneporter, son sistemas de transfección eficientes para incorporar oligonucleótidos modificados en el núcleo de las células ib3.1. Los poliplejos de pei y los lipoplejos de citofectina son internalizados por distintos mecanismos aunque en ambos casos los oligonucleótidos modificados son degradados significativamente en las células ib3.1. El análisis genescan de electroferogramas fluorescentes constituye un sistema fiable, fácil, sensible y seguro para evaluar y cuantificar la degradación intracelular y extracelular de oligonucleótidos marcados con fluorescencia en fluidos biológicos. La tecnología de pcr-ola constituye un sistema fiable y preciso de cuantificación de reparación génica aplicable a modelos celulares heterocigotos. Los fragmentos sfhr pueden actuar como cebador artefactual en las reacciones de pcr y generar un artefacto que da lugar a falsos positivos en la detección de conversión génica. Es indispensable diseñar los cebadores de detección de la modificación génica fuera de la región de homología con los fragmentos sfhr. La corrección génica de mutaciones puntuales mediada por quimeraplastos, oligonucleótidos monocadena y fragmentos sfhr es un proceso ineficiente en las células ib3.1. Será necesario estimular los mecanismos endógenos de reparación génica para incrementar la frecuencia de reparación génica hasta niveles terapéuticos en dichas células.

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. 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. 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. 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. 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.

Our aim was to understand mechanisms responsible for excessive electrolyte loss in the sweat gland and the potential impact on fluid balance during exercise in heat stress conditions. Human physiological testing under exercise/heat stress and immunofluorescence staining of sweat glands from skin biopsies were compared between healthy individuals (with normal and high sweat sodium chloride concentration, [NaCl]) and with cystic fibrosis patients (CF), who exhibit excessively salty sweat due to a defect of Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR). Three novel findings are presented. First, excessively salty sweat may be associated with reduced expression of CFTR in the sweat gland reabsorptive duct of healthy individuals in addition to in those with CF; however, although a link to a CF gene mutation in healthy individuals with high sweat [NaCl] was not demonstrated, the possibility of an undetected CFTR mutation or polymorphism remains to be investigated as an underlying mechanism. Two, CF and healthy individuals with excessively salty sweat respond to moderate dehydration (3% body weight loss during exercise) with an attenuated rise in serum osmolality, greater relative loss in plasma volume, but similar perceived thirst compared to healthy individuals with "normal" sweat [NaCl]. However, individuals with CF respond to rehydration with hypotonic beverage by drinking less ad libitum in response to reduced serum [NaCl], suggesting that thirst-guided fluid replacement may be more appropriate for this population rather than restoring 100% of sweat loss following dehydration as is often recommended in healthy individuals.

Studies of widely diverse species of a protein are a powerful tool to gain information on the structure and function of the protein. We investigated the response of human, pig, shark and killifish cystic fibrosis trans-membrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTRinh-172, GlyH-101, and glibenclamide. In several expression systems, including isolated perfusions of the rectal gland, primary cell cultures of rectal gland tubules and oocyte expression, we observed fundamental differences in the sensitivity to inhibition by these CFTR blockers. We used primarily two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. In oocyte studies, shark CFTR was insensitive to CFTRinh-172 (maximum inhibition 8 ± 1.4% at 20µM), pCFTR was insensitive to Glibenclamide (maximum inhibition 12.8 ± 4.2% at 200µM), and all species were sensitive to GlyH-101 (maximum inhibition with pCFTR of 80.2 ± 3.6% at 20µM). Shark CFTR was completely insensitive to inhibition by CFTRinh-172 in short circuit current experiments (2.5 ± 0.15 % inhibition of chloride secretion) compared to inhibition with GlyH-101 (56.5 ± 6.56 % inhibition of chloride secretion). Perfusion studies confirmed these results. These experiments demonstrate a profound difference in the sensitivity of different CFTR species to inhibition by CFTR blockers. However, the amino acid residues that have been proposed by site directed mutagenesis studies to be responsible for inhibitor binding are uniformly conserved in all four isoforms studied. Therefore, the differences cannot be explained by simply targeting one amino acid for site-directed mutagenesis. Rather, the potency of the inhibitory actions of CFTRinh-172, Gly-H101 and glibenclamide on the CFTR molecule is dictated by the local environment and the three dimensional structure of residues that form the vestibule and the chloride pore.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 168-199). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 168-199). Also available on the Internet.

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Clinical Epidemiology, Department of Public Health Sciences. Title from pdf file main screen (viewed on February 23, 2010). Includes bibliographical references.

The Gram-negative bacterium Pseudomonas aeruginosa is a notorious opportunistic pathogen of individuals with the genetic disease cystic fibrosis (CF). Pseudomonas aeruginosa establishes a chronic infection within the CF lung, where the sputum accumulation characteristic of CF provides a complex and copious growth substrate. P. aeruginosa can grow to high densities in vivo (>10⁹ cells/ml lung sputum), and exacerbations associated with P. aeruginosa high density in vivo growth are primary contributors to CF morbidity and mortality. Surprisingly little is known about the catabolic processes that underlie P. aeruginosa in vivo growth. Unfortunately, nutritional modeling of the CF lung environment in animal models is difficult, as current animal models fail to mimic the sputum accumulation characteristic of CF. In this dissertation, I describe the use of expectorated CF sputum as a P. aeruginosa in vitro growth medium. Using global expression analysis, I show that P. aeruginosa up-regulates genes important for amino acid and lactate metabolism during growth in CF sputum as compared to a laboratory medium. P. aeruginosa also demonstrates enhanced production of the cell-cell communication signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal, PQS), a critical regulator of virulence factor production, during growth in CF sputum. Further, I use chemical analyses of CF sputum samples to develop a defined, synthetic medium that can be used to nutritionally model in vivo conditions. Using this medium, I show that PQS biosynthesis and aromatic amino acid metabolism are intimately linked and that cell-cell communication mediated by PQS is strikingly dependent upon the growth environment of P. aeruginosa. In addition, I demonstrate that P. aeruginosa preferentially consumes specific carbon sources present in the CF sputum milieu during rapid growth. I also describe the use of in vivo-relevant nutrient concentrations to evaluate the potential for P. aeruginosa anaerobic growth in CF sputum. Finally, I describe the purification and characterization of the aromatic amino acid-responsive transcriptional regulator PhhR and discuss its potential role in regulation of P. aeruginosa in vivo carbon substrate preference. / text

The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy