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Cystic fibrosis [keywords]
- Pseudomonas aeruginosa-Induced Apoptosis in Airway Epithelial Cells Is Mediated by Gap Junctional Communication in a JNK-Dependent Manner. [JOURNAL ARTICLE]
- J Immunol 2014 Apr 14.
Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.
- Porcine nasal epithelial cultures for studies of cystic fibrosis sinusitis. [JOURNAL ARTICLE]
- Int Forum Allergy Rhinol 2014 Apr 14.
Transgenic cystic fibrosis (CF) murine models do not develop spontaneous lung or sinus disease, 2 major causes of morbidity in human CF patients. Because of these limitations, transgenic cystic fibrosis transmembrane conductance regulator (CFTR)(-/-) pigs have been developed and are currently being characterized. These CF animal models have phenotypes closely resembling that of human CF subjects. The objectives of the current study were to develop primary porcine nasal epithelial (PNE) cultures and evaluate their usefulness as a means to investigate sinonasal transepithelial transport and CFTR function.PNE derived from the septum or turbinates of CFTR(+/+) and CFTR(-/-) pigs were cultured at an air-liquid interface to confluence and full differentiation. Epithelial monolayers were mounted in Ussing chambers to investigate pharmacologic manipulation of ion transport. Ciliary beat frequency (CBF) and scanning electron microscopy of monolayers were used to indicate degree of ciliation and cell differentiation.Stimulation of CFTR-mediated anion transport (ΔIsc in μA/cm(2) ) was significantly greater in epithelia derived from the septum when compared to turbinates (33.04 ± 1.17 vs 18.9 ± 0.73; p < 0.05). Cyclic adenosine monophosphate (cAMP)-activated Cl(-) secretion was absent in CFTR(-/-) and present in CFTR(+/+) epithelia. Calcium-activated Cl(-) (CaCC) secretion was increased in CF; however, overall Cl(-) transport through CaCCs was very low. Degree of ciliation (90%) and CBF were similar between groups.Septal PNE exhibit a robust ion transport phenotype and indicate CFTR(-/-) sinus disease could be attributable to diminished alternative pathways for Cl(-) transport. Overall, PNE have similarities to human respiratory epithelia not demonstrated in murine cells and represent useful in vitro models for studying CF sinus disease.
- Could T cells be involved in lung deterioration and hyperglycemia in cystic fibrosis? [REVIEW]
- Diabetes Res Clin Pract 2014 Mar 19.
Cystic fibrosis-related diabetes (CFRD) is the most frequent complication of cystic fibrosis (CF) and associated with increased mortality. Why patients have an accelerated loss of lung function before the diagnosis of CFRD remains poorly understood. We reported that patients with or without CFRD had increased glucose excursions when compared to healthy peers. Studies have demonstrated that patients with CF have increased glucose fluctuations and hyperglycemia and that this may affect the clinical course of CF and lead to lymphocyte dysfunction. T-helper 17 (Th17) lymphocytes produce and secrete the pro-inflammatory cytokine IL-17. The Th17 pathway is involved in CF lung inflammation, β-cell destruction in type 1 diabetes (T1D) and Th17 cells of patients with type 2 diabetes have increased production of IL-17 when compared to healthy peers. Also, regulatory T-cells (Tregs) have been shown to be dysfunctional and produce IL-17 in T1D. Furthermore, vitamin D can affect inflammation in CF, diabetes and the differentiation of lymphocytes. In this review, we discuss the potential roles of hyperglycemia on Th17 cells, Tregs and IL-17 as a potential cause for accelerated lung function decline before CFRD and how this could be modulated by vitamin D or by directly intervening in the IL-17A pathway.
- Bile deficiency induces changes of intestinal Cl(-) and HCO3 (-) secretions in mice. [JOURNAL ARTICLE]
- Acta Physiol (Oxf) 2014 Apr 15.
Biliary tract obstruction is a common clinical lesion. However, the effect of biliary tract obstruction on intestinal secretion is poorly understood. In this study, we made an investigation on intestinal HCO3 (-) and Cl(-) secretions in an experimental model of murine biliary duct ligation.Murine intestinal mucosal HCO3 (-) and Cl(-) secretions were examined in vitro in Ussing chambers by pH-stat and short circuit current (Isc ) techniques. The mRNA and protein expressions of the cystic fibrosis transmember conductance regulator (CFTR) and the Na(+) -K(+) -2Cl(-) cotransporter (NKCC1) were analyzed by real time PCR, western blot, and immunohistochemistry.Basal Cl(-) secretion and forskolin-stimulated duodenal and jejunal mucosal HCO3 (-) and Cl(-) secretions in mice with common biliary duct ligation were markedly elevated, compared with controls (P<0.05 and P<0.01). Further experiments showed that basal Cl(-) secretion and forskolin-stimulated duodenal and jejunal mucosal HCO3 (-) and Cl(-) secretions in mice with external bile drainage were also markedly elevated. CFTRinh -172 inhibited forskolin-stimulated HCO3 (-) and Cl(-) secretions. The mRNA and protein expression levels of CFTR and NKCC1 in the intestinal mucosa with both biliary duct ligation and external bile drainage were markedly higher than those in controls (P<0.001). Bile acid administration restored the changes of function and expression of CFTR and NKCC1 in the intestinal mucosa.Bile deficiency in the intestine up-regulates the expressions of intestinal mucosal CFTR and NKCC1, and enhances intestinal mucosal HCO3 (-) and Cl(-) secretion capacity, which contributes to the understanding of intestinal physiologic function for patients with biliary duct obstruction. This article is protected by copyright. All rights reserved.
- The short and long term effects of exercise training in non-cystic fibrosis bronchiectasis - a randomised controlled trial. [JOURNAL ARTICLE]
- Respir Res 2014 Apr 15; 15(1):44.
Exercise training is recommended for non-cystic fibrosis (CF) bronchiectasis, but the long-term effects are unclear. This randomised controlled trial aimed to determine the effects of exercise training and review of airway clearance therapy (ACT) on exercise capacity, health related quality of life (HRQOL) and the incidence of acute exacerbations in people with non-CF bronchiectasis.Participants were randomly allocated to 8 weeks of supervised exercise training and review of ACT, or control. Primary outcomes of exercise capacity and HRQOL (Chronic respiratory disease questionnaire) and secondary outcomes of cough-related QOL (Leicester cough questionnaire) and psychological symptoms (Hospital anxiety and depression scale) were measured at baseline, following completion of the intervention period and at 6 and 12 months follow up. Secondary outcomes of the exacerbation rate and time to first exacerbation were analysed over 12 months.Eighty-five participants (mean FEV1 74% predicted; median Modified Medical Research Council Dyspnoea grade of 1 (IQR [1-3]) were included. Exercise training increased the incremental shuttle walk distance (mean difference to control 62 m, 95% CI 24 to 101 m) and the 6-minute walking distance (mean difference to control 41 m, 95% CI 19 to 63 m), but these improvements were not sustained at 6 or 12 months. Exercise training reduced dyspnoea (p = 0.009) and fatigue (p = 0.01) but did not impact on cough-related QOL or mood. Exercise training reduced the frequency of acute exacerbations (median 1[IQR 1-3]) compared to the control group (2[1-3]) over 12 months follow up (p = 0.012), with a longer time to first exacerbation with exercise training of 8 months (95% CI 7 to 9 months) compared to the control group (6 months [95% CI 5 to 7 months], p = 0.047).Exercise training in bronchiectasis is associated with short term improvement in exercise capacity, dyspnoea and fatigue and fewer exacerbations over 12 months.Trial registry: ClinicalTrials.gov(NCT00885521).
- [Bacteria of the Burkholderia cepacia complex: epidemiology and diagnosis of infection in patients with cystic fibrosis]. [English Abstract, Journal Article]
- Epidemiol Mikrobiol Imunol 2014 Apr; 63(1):18-26.
Bacteria of the Burkholderia cepacia complex are the cause of severe lung infections primarily in patients with cystic fibrosis (CF). The risk of epidemic spread of the pathogen in the community of CF patients was the reason for implementing strict isolation measures and identifying the bacterium as early as possible. This review article features the taxonomy of the Burkholderia cepacia complex, possibilities for the laboratory diagnosis of all representatives of this complex, and epidemiological situation in the communities of CF patients in the Czech Republic and in the world. Keywords: Burkholderia cepacia complex - cystic fibrosis - epidemic strain - pathogenesis of the infection.
- A Burkholderia cenocepacia gene encoding a non-functional tyrosine phosphatase is required for the delayed maturation of the bacteria-containing vacuoles in macrophages. [JOURNAL ARTICLE]
- Microbiology 2014 Apr 12.
Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles (BcCVs) that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low-molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the BCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since BCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in B. multivorans confers to this bacterium a similar phagosomal maturation delay as found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity.
- LMTK2 Mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells. [JOURNAL ARTICLE]
- J Biol Chem 2014 Apr 11.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a Cl--selective ion channel expressed in fluid-transporting epithelia. Lemur Tyrosine Kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-S737 in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl- secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared to the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-S737 mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl- channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl- channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.
- Understanding how cystic fibrosis mutations disrupt CFTR function: From single molecules to animal models. [REVIEW]
- Int J Biochem Cell Biol 2014 Apr 10.
Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2,000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised.Studiesof a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients.
- Asymmetric Dimethylarginine in Chronic Obstructive Pulmonary Disease (ADMA in COPD). [Journal Article]
- Int J Mol Sci 2014; 15(4):6062-71.
l-Arginine metabolism including the nitric oxide (NO) synthase and arginase pathways is important in the maintenance of airways function. We have previously reported that accumulation of asymmetric dimethylarginine (ADMA) in airways, resulting in changes in l-arginine metabolism, contributes to airways obstruction in asthma and cystic fibrosis. Herein, we assessed l-arginine metabolism in airways of patients with chronic obstructive pulmonary disease (COPD). Lung function testing, measurement of fractional exhaled NO (FeNO) and sputum NO metabolites, as well as quantification of l-arginine metabolites (l-arginine, l-ornithine, l-citrulline, ADMA and symmetric dimethylarginine) using liquid chromatography-mass spectrometry (LC-MS) were performed. Concentrations of l-ornithine, the product of arginase activity, correlated directly with l-arginine and ADMA sputum concentrations. FeNO correlated directly with pre- and post-bronchodilator forced expiratory volume in one second (FEV1). Sputum arginase activity correlated inversely with total NO metabolite (NOx) and nitrite concentrations in sputum, and with pre- and post-bronchodilator FEV1. These findings suggest that ADMA in COPD airways results in a functionally relevant shift of l-arginine breakdown by the NO synthases towards the arginase pathway, which contributes to airway obstruction in these patients.