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- Hussain, Rashida, 1976-
(författare)
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Cell responses in infected and cystic fibrosis respiratory epithelium
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Respiratory Epithelium. Örebro Studies in Medicine 99. Cystic fibrosis (CF) is caused by a mutation in a cAMP-activated chloride (Cl-) channel (CFTR). Mortality and morbidity in CF is mainly due to the deregulated responses of the airway epithelial cells. The purpose of the thesis was to investigate the behaviour of the airway epithelial cells that are involved in maintaining the homeostasis in the airways.Nasal brush biopsies obtained from anesthetized human nasal mucosa can be an easy source to establish primary epithelial cell lines (Paper I). We found that CF and non CF cellular models cannot fully show the relation between CFTR and the phenotypic differences between CF and healthy cells (Paper II). The possibility to correct the Cl- transport defect in CF by the use of stable NOdonors, and ambroxol was investigated. NO-donors stimulated Cl- efflux, and decreased ENaC mRNA expression in CFBE cells (Paper III), while ambroxol increased Cl- efflux from CFBE cells, and showed a positive effect on the biosynthesis of CFTR (Paper IV). This suggests that these substances may be a potentially interesting group of compounds for the treatment of CF. Increased levels of IL-6 and IL-8 upon infection in CF cells can increase the susceptibility of P. aeruginosa infected CF cells to apoptosis and/or internalization of these bacteria in CF cells and hence, may have important roles in the pathology of P. aeruginosa infection in CF airways. If internalization is beneficial for the host then glucocorticoids (GCs) are not beneficial for the treatment of CF patients. However, GCs may improve airway hydration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out (Paper V). The neonatal isolates S. epidermidis 94B080 and S. aureus 90B083 can modulate CFTR and ENaC expression in airway epithelial cells, which may disturb the ion transport in the respiratory epithelium upon bacterial exposure. Airway epithelial cells also show excessive inflammatory responses to these bacteria, which means that these bacteria may induce pulmonary inflammation (Paper VI).
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| 2. |
- Oliynyk, Igor
(författare)
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Advances in Pharmacological Treatment of Cystic Fibrosis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cystic fibrosis (CF) is an inborn, hereditary disease, due to mutations in the gene for a cAMP-activated chloride (Cl-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). As a result of impaired ion and water transport,the airway mucus is abnormally viscous, which leads to bacterial colonization.Recurrent infections and inflammation result in obstructive pulmonary disease.Similar changes in the pancreas lead to pancreatic insufficiency.Several compounds have been tested to improve transepithelial ion transport in CF patients, either via activation of the mutant CFTR, or via stimulation of alternative chloride channels. The main purpose of this thesis was to find substances that might correct the defective ion transport in epithelial cells in CFand could be useful for the pharmacological treatment of CF patients. Long-term treatment with the macrolide antibiotic azithromycin (AZM)improved clinical parameters and lung function in CF patients and increased Cl- transport in CF bronchial epithelial cells (CFBE) (Paper I); although mRNA expression of the CFTR gene remained unchanged.In contrast, pre-exposure to the mucolytic antioxidant N-acetylcysteine (NAC) increased CFTR protein expression and was associated with increased Cl- efflux from CFBE cells (Paper II). Clinical trials of this substance might be warranted. Duramycin has been the subject of clinical trials that finished in June2009. Up till now, no results from this study are available. The effect of this substance on Cl- efflux from three CF and three non-CF cell lines (Paper III) was disappointing. An effect was found only in CFBE cells, the effect was minimal, occurred in a narrow concentration range, and was not associated with an increase in the intracellular calcium concentration [Ca2+]i. The fact that NO-donors stimulated Cl- efflux from CFBE cells (but did notchange [Ca2+]i) after several hours of preincubation suggests that these substances may be a potentially interesting group of compounds for the treatment of CF (Paper IV). A model for the effect of NO-donors on Cl- efflux is presented.
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| 3. |
- Dragomir, Anca, 1974-
(författare)
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Approaches to Pharmacological Treatment and Gene Therapy of Cystic Fibrosis
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest.Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies.Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients.Successful in vitro transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.
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| 4. |
- Nilsson, Harriet
(författare)
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Studies of Tight Junctions and Airway Surface Liquid in Airway Epithelium with Relevance to Cystic Fibrosis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cystic fibrosis (CF) is a multi-organ autosomal recessive disease of fluid-transporting epithelia, due to a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is a cAMP-regulated Cl-channel involved in various regulatory processes. Salt and water transport depend on CFTR and the epithelial sodium channel (ENaC), operating in concert with the paracellular pathway through the tight junctions (TJ). The ionic composition of the ASL has been assumed to be altered in CF, resulting in a fatal accumulation of viscous mucus in the airways. ASL samples were collected from tracheal and nasal fluid in normal and transgenic CF mice and from the fluid covering the apical surface of normal bronchial cells (16HBE14o-) and a CF human bronchial cell line (CFBE41o-). Analysis of the elemental content of the ASL was performed by X-ray microanalysis. The ASL contained more Na and Cl in CFTR-deficient or DF508-CFTR-containing cells than in control cells with wild- type CFTR. The relation between osmolarity and TJ permeability was examined by the addition of salt or sugar (295-700 mOsm) to 16HBE14o- cells, where the integrity of TJ was evaluated by transepithelial electrical resistance (TEER) measurements. Studies of interaction between the activity of CFTR, TJ and cytoskeleton were performed in CFBE41o-, plasmid corrected CFBE41o- (CFBE41o pCep4), and 16HBE14o- cells exposed to an inhibitor of CFTR (CFTRinh-172). The TJ were investigated by determining the paracellular permeability to lanthanum ions or with [14C] mannitol. Cytoskeletal changes were evaluated by immunofluorescence. Hyperosmotic stress resulted in opening of TJ. Inhalation of hypertonic salt or sugar solutions may open TJ, leading to enhanced paracellular water transport and increased ASL volume, diluted mucus and enhanced mucociliary clearance. This may explain the beneficial effect of this treatment for CF-patients. In healthy airway epithelial cells, inhibition of CFTR by CFTRinh-172 resulted in an increased TEER, whereas stimulation of CFTR by IBMX/forskolin caused a decrease. The paracellular permeability was inversely proportional to TEER. Immunofluorescence revealed a disorganization of cytoskeletal proteins in CF-cells. These results point toward a possible interaction between the activity of CFTR and TJ protein complex, presumably via the cytoskeleton.
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