A Fibrose cística é uma doença autossômica recessiva que tipicamente se manifesta por doença pulmonar obstrutiva crônica, insuficiência exócrina do pâncreas e elevada concentração de eletrólitos no suor. É causada por mutações no gene Cystic Fibrosis Transmembrane Regulator (CFTR). A disfunção da CFTR causa obstrução principalmente das vias aéreas e dos ductos pancreáticos. O curso e a gravidade da manifestação pulmonar não estão sempre diretamente relacionados ao genótipo especifico da CFTR, sugerindo uma forte ação de genes modificadores ou de variantes intragênicas, que podem influenciar na gravidade do fenótipo associado. Objetivo: Este estudo propôs caracterizar clínica e geneticamente uma amostra de pacientes com fibrose cística no Hospital Universitário João de Barros Barreto (Pará, Brasil) e estabelecer uma possível associação da variante p.M470V com a gravidade da doença respiratória. Metodologia: Um questionário clínico e epidemiológico foi aplicado para coleta de dados dos pacientes em dois momentos distintos. O DNA foi extraído e realizado seqüenciamento direto dos éxons: 11, 12, 18, 19, 21 e 22 utilizando sequenciador automático ABI Prism – 3130 (Applied Biosystems). Resultados: Foram coletados dados clínicos de 135 pacientes, porém o estudo genético só foi possível em 125 destes. A média de idade dos pacientes estudados foi de 15,44 ± 11,8 anos (mediana: 13 anos) e ao diagnóstico foi de 10,00 ± 9,6 anos (mediana: 7 anos), onde 56,3% eram do sexo masculino e 82,2% pardos. Ao diagnóstico 94,8% dos pacientes eram sintomáticos: respiratórios (92,6%), digestivos (34,9%) e 32,6% desnutridos. Pseudomonas aeruginosa (37,4%) e Staphylococcus aureus (49,63%) foram os microorganismos mais frequentes do trato respiratório. O éxon 11 foi o que apresentou uma maior prevalência de alterações patogênicas e não patogênicas. A frequência alélica da mutação p.F508del foi 14,8% e da p.M470V foi 56,4%. O estudo não demonstrou associação de p.M470V isoladamente com gravidade de doença respiratória, porém a mutação p.F508del mostrou associação com pior evolução radiológica e infecção por P aeruginosa. Conclusão: O estudo revelou um diagnóstico tardio nesta amostra, e não esclareceu o papel da variante p.M470V na evolução da doença respiratória da FC. / Cystic fibrosis is an autosomal recessive disease that typically manifests as chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and a high concentration of electrolytes in sweat caused by mutations in the gene Cystic Fibrosis Transmembrane Regulator (CFTR). The dysfunction of CFTR causes obstruction, especially of the airways and pancreatic ducts. The course and severity of pulmonary manifestations are not always directly related to the specific CFTR genotype, suggesting a strong action of modifying genes and of intragenic variants, which may influence the severity of the associated phenotype. Objective: This study aimed to clinically and genetically characterize a sample of patients with cystic fibrosis in the Hospital Universitário João de Barros Barreto (Pará, Brazil) and establish a possible association of the variant p.M470V with the severity of respiratory disease. Methods: A clinical and epidemiological questionnaire was applied to collect data from patients two different times. DNA was extracted and direct sequencing of exons 11 , 12 , 18 , 19 , 21 and 22 was performed using an automated sequencer ABI Prism - 3130 (Applied Biosystems). Results: Clinical data of 135 patients were collected; however, the genetic study was only possible in 125 of these. The mean age of patients was 15.44 ± 11.8 years (median: 13 years) and, at diagnosis, was 10.00 ± 9.6 years (median: 7 years), where 56.3% were male, the majority brown (82.2%). At diagnosis 94.8% of patients were symptomatic: respiratory (92.6%) , digestive (34.9%) ,and 32.6% malnourished. Pseudomonas aeruginosa (37.4%) and Staphylococcus aureus (49.63%) were the most frequent microorganisms of the respiratory tract. Exon 11 accounted for the majority of the variants found. The allelic frequency of p.508del mutation was 14.8% and of p.M470V was 56.4%. The study showed no association of p.M470V with severity of respiratory disease, but the p.F508del mutation was associated with worsening radiological evolution and P aeruginosa infection. Conclusion: The study revealed a delayed diagnosis in this sample, and did not clarify the role of p.M470V in respiratory disease.

No description available.

Ionenbewegungen über das Atemwegsepithel bilden die Grundlage einer definierten Zusammensetzung intraluminaler pulmonaler Flüssigkeit. Ein perinataler Anstieg maternaler und damit einhergehend fetaler Glukokortikoid (GC)-Serumlevel ist vergesellschaftet mit einer Veränderung im epithelialen Ionentransport der Lunge. So erfährt das respiratorische Epithel in einer perinatalen Anpassungsreaktion eine Transition fetaler Fruchtwasserproduktion hin zu überwiegend flüssigkeitsabsorbierenden Vorgängen. In den distalen Abschnitten spiegelt sich dies in einer Aktivierung flüssigkeitsabsorptiv wirkender epithelialer Natriumkanäle (ENaC) wider. Vor Beginn der Transition überwiegt ein sekretorisch aktiver, apikaler Chloridkanal, der cystic fibrosis transmembrane conductance regulator (CFTR). Als Gegenspieler zum ENaC gewährleistet der CFTR einen transepithelialen Flüssigkeitsstrom in das Lumen des respiratorischen Systems. Diese Prozesse ermöglichen den pulmonalen Gastaustausch sowie die Bereitstellung eines periziliären Flüssigkeitsfilms, welcher die postnatale mukoziliäre Clearance (MCC) gewährleistet. Während bereits gezeigt werden konnte, dass GC den ENaC vor allem in distalen Atemwegsepithelien in Expression und Aktivität steigern (Thome et al. 2003; Venkatesh und Katzberg 1997), gibt es bisher nur wenige Daten zum Einfluss von GC auf den CFTR. Zusammenfassend kann durch die vorliegende Arbeit gezeigt werden, dass es durch GC zu einer Zunahme des CFTR-abhängigen Ionentransportes kommt und dass dieser aktivitätssteigernde Einfluss von der PI3K sowie der AKT abhängig ist, während die SGK1 nicht wesentlich in diesen Wirkmechanismus involviert zu sein scheint. Es ließ sich nachweisen, dass es durch GC zu einer gesteigerten Phosphorylierung von AKT, SGK1 sowie NEDD4L kommt, woraus eine Aktivitätssteigerung der AKT und der SGK1 sowie ein Rückgang in der NEDD4L-Aktivität resultieren. Eine AKT-Inhibition verhindert die zunehmende NEDD4L Phosphorylierung, während eine Inhibition der SGK1 keinen Einfluss auf den NEDD4L-Phosphorylierungsstatus zeigt. Des Weiteren ergibt sich ein stimulierender Einfluss von GC auf die CFTR-Aktivität innerhalb von 30 Minuten. Im Gegensatz zum Ionentransport zeigten GC keinen Einfluss auf den Epithelwiderstand und damit die Barrierefunktion der Zellkultur.:1. Abkürzungsverzeichnis S. 3 2. Abbildungsverzeichnis S. 5 3. Bibliographische Beschreibung S. 6 4. Einleitung S. 7 4.1. Der CFTR und seine Rolle im epithelialen Chloridtransport S. 7 4.2.Die Struktur und Regulation des CFTR S. 8 4.3.Die (Patho-)Physiologie des CFTR S. 10 4.4.Alternative Chloridkanäle S. 11 4.5.Methodik – Etablierung der Methode S. 12 5. Originalpublikation 5.1. “Glucocorticoids Distinctively Modulate the CFTR Channel with Possible Implications in Lung Development and Transition into Extrauterine Life” S. 14 5.2. “Signaling Cascade involved in Rapid Stimulation of Cystic Fibrosis Conductance Regulator (CFTR) by Dexamethasone” S. 36 6. Zusammenfassung der Ergebnisse S. 52 6.1 Hintergrund und Ziel S. 52 6.2 Ergebnisse S. 53 7. Diskussion der Ergebnisse S. 54 8. Literaturverzeichnis S. 57 9. Anmerkungen zur Originalpublikation – Darstellung des eigenen Beitrags S. 64 10. Erklärung über die eigenständige Abfassung der Arbeit S. 65 11. Curriculum vitae S. 66 12. Danksagung S. 68

The genetic disease cystic fibrosis is the most common lethal genetic disease in Western countries. People born with cystic fibrosis suffer from many health issues including severe respiratory problems, inflammation and recurrent lung infections that can become fatal. The disease is caused by the loss of function of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an chloride ion channel and, in healthy people, its activity assures correct water and salt transport across the cell membrane. Most cases of cystic fibrosis are caused by a genetic defect that leads to the deletion of phenylalanine 508 (F508del) in the amino acid sequence of the protein. The molecular mechanism by which F508del leads to loss of function of the CFTR channel is still poorly understood. The mutation is found in the first nucleotide binding domain (NBD1) and studies have shown that it causes misfolding of CFTR and subsequent degradation of the protein by the cellular quality control system. It is established that the mutation affects stability and dynamics of NBD1 but does not alter its structure significantly. This destabilizing effect of F508del can be compensated by specific mutations distributed over different regions of NBD1, leading to recovery of membrane expression of a functional channel. A surprising example involves the regulatory insertion (RI), a 32-residue long segment found in all CFTR orthologs but not in related channels or transporters. The RI is not resolved in crystal structures of NBD1 nor cryo-EM structures of CFTR and has been described as intrinsically disordered. Its functional role in CFTR is unknown. Removal of the RI increases the stability of the NBD1 domain and, in the context of F508del-CFTR, this deletion restores maturation, cell surface expression and activity of the mutant channel. We probed the effect of the RI on NBD1 structure, dynamics and allostery using X-ray crystallography, single molecule FRET and hydrogen-deuterium exchange. We discovered that the RI enables an alternative NBD1 fold which departs markedly from the canonical fold previously observed for this domain and the NBDs of other ABC transporters. The conformational equilibrium between these states is regulated by ATP binding and affected by disease-associated conditions. Aside from clear alterations to structure and dynamics of NBD1, the RI also affects allostery, i.e. how NBD1 structure and dynamics respond to perturbations such as ligand binding. Finally, we show that the RI-enabled conformation is adopted in full-length CFTR and associated with increased channel activity in electrophysiological assays. We then identify an allosteric network that links the structural hotspots of the conformational changes to F508 and its surroundings. Lastly, we argue that these conformational changes lead to unfolding of NBD1 in the context of F508del, providing a new model for the molecular mechanism leading to pathogenesis. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No description available.

In cystic fibrosis (CF) therapy, the recent approval of CF-transmembrane conductance regulator (CFTR) channel modulators is considered to be the major breakthrough. However, the current first-line approach based mainly on pulmonary function to measure effects of the novel therapy, tested by forced expiratory volumes in one second (FEV1), provides restricted sensitivity to detect early structural damages. Accordingly, there is a need for new sensitive surrogate parameters. Most interestingly, these should quantify inflammation that precedes a decline of pulmonary function. We present a novel method assessing inflammatory markers in the upper airways’ epithelial lining fluid (ELF) obtained by nasal lavage (NL). In contrast to broncho-alveolar lavage, ELF sampling by NL is an attractive method due to its limited invasiveness which allows repeated analyses, even performed in a home-based setting. In a longitudinal cohort study (ClinicalTrials.gov, Identifier: NCT02311140), we assessed changes of inflammatory mediators in 259 serially obtained nasal lavages taken up to every second day before and during therapy with ivacaftor from ten CF patients carrying a G551D mutation. Patients were trained to sample NL-fluid at home, to immediately freeze and transfer chilled secretions to centers. Neutrophil Elastase, Interleukins IL-1b, IL-6 and IL-8 in NL were quantified. During 8-12 weeks of ivacaftor-treatment, median values of IL-1b and IL-6 significantly declined 2.29-fold (2.97!1.30 pg/mL), and 1.13-fold (6.48!5.72 pg/mL), respectively. In parallel, sweat tests and pulmonary function improved considerably. This is the first study assessing changes of airway inflammation on a day-to-day basis in CF patients receiving a newly administered CFTR-modulator therapy. It proves a decline of airway inflammation during ivacaftor-therapy.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder involving a mutation in the CF transmembrane conductance regulator protein (CFTR), which causes dysfunctional transport of chloride ions across cell membranes. CF affects multiple body systems and a few of its symptoms include chronic cough, difficulty breathing, obstructive airway disease, bacterial pulmonary infections, maldigestion, malabsorption, pancreatitis, and male infertility. Until recently, treatment options have been limited to alleviating symptoms, but a new classification of drugs, CFTR modulators, provide an opportunity to slow the progression of the disease and improve clinical outcomes. The effect of CFTR modulators may be attributed to the reduction of persistently colonizing bacteria in CF lungs. Though, the effects of modulators on microbial communities colonizing the CF lung remains unknown, specifically with common respiratory pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Particularly, previous CF studies have been limited in scope due to focusing on only one type of modulator and by using low-yield sequencing techniques. To address this gap, we seek to study the changes in CF respiratory pathogens of patients initiating CFTR modulator therapy at Nemours Hospital using long-read metagenomic sequencing (Oxford Nanopore) of longitudinally collected respiratory samples. We have optimized a protocol for host DNA depletion and microbial metagenomic sequencing to characterize the respiratory microbiome. This study focuses on utilizing these sequencing data to compare the microbiome among two healthy controls to pre-CFTR-treatment microbial communities of two recruited pediatric CF patients.

No description available.

No description available.

No description available.