To Örebro University

Conclusion: Using a local anesthetic agent before obtaining nasal biopsies by nasal brushing makes the sampling procedure smooth, avoids lacrimation, nasal itching/irritation, and/or sneezing and provides enough viable cells to establish primary cultures.

Objectives: To examine the use of local anesthesia to avoid the irritation experienced by the subject when nasal biopsies are obtained by nasal brushing in order to culture viable nasal epithelial cells.

Methods: Nasal epithelial cells were collected from the mid-part of the inferior turbinate of healthy volunteers by brushing with interdental brushes, after spraying a topical anesthetic on the nasal mucosa. Immunocytochemistry was performed to assess the purity of epithelial cells.

Results: Cell samples ranging from 1.16 x 10(5) to 3.06 x 10(5) cells/per sample were obtained. Of 11 samples, 7 formed confluent cultures, while the remaining 4 samples showed only patches of epithelial cells. Neither fungal nor bacterial contamination posed a problem. Immunocytochemistry of the cytospin slides confirmed the presence of epithelial cells in the cultures. No adverse effects were experienced by the volunteers.

with endogenous wild-type cystic fibrosis transmembrane conductance regulator (CFTR), CFBE cells with mutated ΔF508-CFTR, corrected CFBE cells overexpressing CFTR, CFSME (CF submucosal) and Calu-3 (non-CF submucosal) cells with respect to the epithelial sodium channel (ENaC), inducible NO synthase (iNOS), and mucins (MUC) (studied by quantitative Real-Time-Polymerase Chain Reaction, qRT-PCR, and Western blot), and wound healing.

CFBE cells had significantly more expression of b- and g-ENaC mRNA and of b-ENaC protein than 16HBE cells. Compared to corrected CFBE cells, CFBE cells had increased mRNA expression of all ENaC subunits and b-ENaC protein. For ENaC, the CFSME/Calu-3 mRNA ratio was very low and contradictory to the ENaC upregulation in CF cells. CFBE cells showed decreased expression of iNOS at both mRNA and protein levels compared to 16HBE cells and only at the mRNA level compared to corrected CFBE cells. CFSME cells showed expression of iNOS whereas Calu-3 cells did not. Higher expression of MUC2 and MUC5B was found in corrected CFBE cells compared to CFBE cells. Wound healing in CFBE cells was delayed compared to corrected CFBE cells, but not to 16HBE cells, and in CFSME cells compared to Calu-3 cells.

Our data suggest CFSME as an inappropriate CF cell model for Calu-3 cells, and provide only partial support for the theory that the differences (in ENaC, iNOS, mucins, and wound healing) between these cell lines are associated to the presence of CFTR in the bronchial airway epithelial cells.

Since previous studies showed that the endogenous bronchodilator, S nitrosglutathione (GSNO), caused amarked increase in CFTR-mediated chloride (Cl−) efflux and improved the trafficking of CFTR to the plasmamembrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl− efflux, itwas investigatedwhether the NO-donor properties of GSNOwere relevant for its effect on Cl− efflux fromairwayepithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-Nacetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitricoxide adduct (DEA-NONOate) on Cl− efflux from CFBE (ΔF508/ΔF508-CFTR) airway epithelial cells was tested.Cl− efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide(MQAE)-technique. Possible changes in the intracellular Ca2+ concentration were tested by the fluorescent fluo-4method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, anincreased Cl− efflux was found (in the order SNAP > DETA-NO > SNP). The effect of DEA-NONOate on Cl− effluxwas not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as withGSNO, after a short (5 min) incubation, SNP had no significant effect on Cl− efflux. None of the NO-donors thathad a significant effect on Cl− efflux caused significant changes in the intracellular Ca2+ concentration. After 4 hpreincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOatedecreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only inα-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on theexpression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concludedthat the effect of GSNO on Cl−efflux is, at least in part, due to its properties as an NO-donor, and the effect is likely tobe mediated by CFTR, not by Ca2+-activated Cl− channels.

Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratoryepitheliumis covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl⁻ efflux viathe cystic fibrosis transmembrane conductance regulator (CFTR) and Naþ influx via the epithelial Naþ channel (ENaC). In cellsexpressing wt-CFTR, ambroxol increased the Cl- conductance, but not the bicarbonate conductance of the CFTR channels.Weinvestigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airwayepithelial cells (CFBE) cells. CFBE cells were treated with 100 mM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR andENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Westernblot. The effect of ambroxol on Cl− transport was measured by Cl− efflux measurements with a fluorescent chloride probe.Ambroxol significantly stimulated Cl− efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced theexpression of the mRNA of CFTR and a-ENaC, and of the CFTR protein. No significant difference was observed in b-ENaCafter exposure to ambroxol, whereasmRNA expression of g-ENaC was reduced. No significant effects of ambroxol on the ENaCsubunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca²⁺ concentration.Upregulation of CFTR and enhanced Cl⁻ efflux after ambroxol treatment should promote transepithelial ion and watertransport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients.

Pseudomonas aeruginosa infection is common in cystic fibrosis (CF). Uptake of P. aeruginosa by the cell and the subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. CF airways have elevated levels of IL-6 and IL-8. Glucocorticoids (GCs) are anti-inflammatory but their use in CF is controversial. We studied the effect of IL- 6, IL-8 and GCs on bacterial internalization, apoptosis, and intracellular Ca2+concentration in CF bronchial epithelial (CFBE) cells and found that increased levels of IL-6 and IL-8 can increase the susceptibility of P. aeruginosa infected cells to apoptosis and/or internalization of these bacteria in CF cells. GCs decreased the extent of apoptosis in CFBE cells infected with P. aeruginosa, but may improve airway hydration by increasing the intracellular Ca2+ concentration. None of the GCs and cytokines affected apoptosis in cells not exposed to Pseudomonas. We conclude that increased levels of IL-6 and IL-8 may have important roles in the pathology of P. aeruginosa infection in CF airways. If internalization is beneficial for the host then GCs are not beneficial for the treatment of CF patients. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out. 

Bacteria affect the respiratory epithelium, which is covered by airway surface liquid (ASL) and mucus. Ion concentrations in the ASL are determined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC). Neonatal sepsis is a major risk factor for subsequent pulmonary disease in preterm newborns. Predominating are coagulase-negative staphylococci (e.g., Staphylococccus epidermidis and Staphylococccus aureus). The aim of this study was to investigate modulation of CFTR, ENaC, mucins, proinflammatory cytokines, and inducible nitric oxide synthase (iNOS) in respiratory epithelial cells after S. epidermidis 94B080 and S. aureus 90B083 exposure. Bronchial epithelial cells were incubated with S. epidermidis 94B080 and S. aureus 90B083 (neonatal blood isolates) for 1-36h. Expression of CFTR, ENaC, iNOS, and mucins was analyzed by real-time PCR and Western blotting. Release of cytokines was analyzed by ELISA, and production of NO by the Griess assay. Expression of CFTR significantly decreased after 36h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of - and -ENaC. Expression of iNOS increased, but NO was not detected. Both staphylococci caused a decrease in the intracellular Ca2+ concentration. S. aureus induced increased secretion of IL-6, IL-8, and transforming nuclear factor (TNF)- in a time-dependent manner as compared with S. epidermidis. In conclusion, expression of ENaC, CFTR, and iNOS is modulated by exposure to S. aureus 90B083 and S. epidermidis 94B080. S. aureus is more potent in causing release of IL-6, IL-8, and TNF- by bronchial epithelial cells as compared with S. epidermidis. The mRNA expression for the mucus proteins MUC2, MUC5AC, and MUC5B could not be measured, neither in the presence nor in the absence of bacteria.