oru.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Cell responses in infected and cystic fibrosis respiratory epithelium
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.ORCID iD: 0000-0003-4946-3228
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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).

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2014. , p. 85
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 99
Keywords [en]
airway epithelial cells, cystic fibrosis, bacterial infection, CFTR, ENaC, chloride transport, intracellular calcium, P. aeruginosa internalization
National Category
Biomedical Laboratory Science/Technology
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-32191ISBN: 978-91-7668-984-4 (print)OAI: oai:DiVA.org:oru-32191DiVA, id: diva2:660282
Public defence
2014-01-17, Bohmansalen, B-huset, Universitetssjukhuset i Örebro, S Grev Rosengatan 18, 703 62 Örebro, 11:57 (Swedish)
Opponent
Supervisors
Available from: 2013-10-29 Created: 2013-10-29 Last updated: 2017-10-17Bibliographically approved
List of papers
1. Isolation and culture of primary human nasal epithelial cells from anesthetized nasal epithelia
Open this publication in new window or tab >>Isolation and culture of primary human nasal epithelial cells from anesthetized nasal epithelia
2014 (English)In: Acta Oto-Laryngologica, ISSN 0001-6489, E-ISSN 1651-2251, Vol. 134, no 3, p. 296-299Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
London, United Kingdom: Informa Healthcare, 2014
Keywords
Nasal brush biopsy, airway epithelia, lidocaine
National Category
Medical and Health Sciences Otorhinolaryngology
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-34502 (URN)10.3109/00016489.2013.859396 (DOI)000331829700014 ()24359095 (PubMedID)2-s2.0-84894384458 (Scopus ID)
Funder
Swedish Heart Lung FoundationSwedish Research Council
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2018-06-05Bibliographically approved
2. ENaC, iNOS, mucins expression, and wound healing in cystic fibrosis airway epithelial and submucosal cells
Open this publication in new window or tab >>ENaC, iNOS, mucins expression, and wound healing in cystic fibrosis airway epithelial and submucosal cells
2013 (English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keywords
Cystic fibrosis, CFTR, ENaC, iNOS, mucin, wound healing, CFTR inh-172
National Category
Medical and Health Sciences
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-32765 (URN)
Funder
Swedish Heart Lung Foundation
Available from: 2013-12-13 Created: 2013-12-13 Last updated: 2018-04-23Bibliographically approved
3. The effect of NO-donors on chloride efflux, intracellular Ca2+ concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells
Open this publication in new window or tab >>The effect of NO-donors on chloride efflux, intracellular Ca2+ concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells
Show others...
2013 (English)In: Experimental and molecular pathology (Print), ISSN 0014-4800, E-ISSN 1096-0945, Vol. 94, no 3, p. 474-480Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Maryland Heights, USA: Elsevier, 2013
Keywords
Nitric oxide donors, Cystic fibrosis, Airway epithelium, CFTR, ENaC, Calcium
National Category
Medical and Health Sciences Cell Biology
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-32767 (URN)10.1016/j.yexmp.2013.03.003 (DOI)000319535900008 ()23523754 (PubMedID)2-s2.0-84876344605 (Scopus ID)
Funder
Swedish Heart Lung FoundationNIH (National Institute of Health)Swedish Research Council
Available from: 2013-12-13 Created: 2013-12-13 Last updated: 2017-12-06Bibliographically approved
4. The effect of ambroxol on chloride transport, CFTR and ENaC in cysticfibrosis airway epithelial cells
Open this publication in new window or tab >>The effect of ambroxol on chloride transport, CFTR and ENaC in cysticfibrosis airway epithelial cells
Show others...
2013 (English)In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 37, no 11, p. 1149-1156Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Hoboken, USA: Wiley-Blackwell, 2013
Keywords
Airway epithelium, ambroxol, cystic fibrosis, Cl− efflux, CFTR; ENaC
National Category
Medical and Health Sciences Cell Biology
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-32769 (URN)10.1002/cbin.10146 (DOI)000325489500002 ()23765701 (PubMedID)2-s2.0-84885861654 (Scopus ID)
External cooperation:
Funder
Swedish Heart Lung FoundationSwedish Research Council
Note

Funding Agencies:

Swedish Science Research Council 

Available from: 2013-12-13 Created: 2013-12-13 Last updated: 2017-12-06Bibliographically approved
5. Effect of IL-6, IL-8 and glucocorticoids on the internalization of Pseudomonas aeruginosa (ATCC 27853) in cystic fibrosis bronchial epithelial cells
Open this publication in new window or tab >>Effect of IL-6, IL-8 and glucocorticoids on the internalization of Pseudomonas aeruginosa (ATCC 27853) in cystic fibrosis bronchial epithelial cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

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. 

Keywords
cystic fibrosis, Pseudomonas aeruginosa, internalization, apoptosis, glucocorticoids.
National Category
Pharmacology and Toxicology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-35865 (URN)
Available from: 2014-08-05 Created: 2014-08-05 Last updated: 2018-01-11Bibliographically approved
6. Modulation of ENaC, CFTR, and iNOS expression in bronchial epithelial cells after stimulation with Staphylococcus epidermidis (94B080) and Staphylococcus aureus (90B083)
Open this publication in new window or tab >>Modulation of ENaC, CFTR, and iNOS expression in bronchial epithelial cells after stimulation with Staphylococcus epidermidis (94B080) and Staphylococcus aureus (90B083)
2013 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 121, no 9, p. 814-826Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Hoboken, USA: Wiley-Blackwell, 2013
Keywords
Airway epithelium, Staphylococcus aureus 90B083, Staphylococcus epidermidis 94B080, CFTR, ENaC, iNOS, cytokines
National Category
Medical and Health Sciences Immunology
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-30514 (URN)10.1111/apm.12138 (DOI)000322757000003 ()23879620 (PubMedID)2-s2.0-84881558007 (Scopus ID)
Note

Funding Agencies:

Nyckelfonden of Örebro University Hospital 

Swedish Science Research Council 

Swedish Heart Lung Foundation 

Available from: 2013-08-30 Created: 2013-08-30 Last updated: 2018-05-21Bibliographically approved

Open Access in DiVA

Cover(115 kB)62 downloads
File information
File name COVER01.pdfFile size 115 kBChecksum SHA-512
d930555cf0c720e238cecf062e415ed33f19a3347506d0f403b3ac2e23baa3005e487cb4383e27baf24f0832cb80a8b5cbe2b448b32a57ecb00565ac8e4ecc27
Type coverMimetype application/pdf
Spikblad(114 kB)88 downloads
File information
File name SPIKBLAD01.pdfFile size 114 kBChecksum SHA-512
bce342e553017063ee62d6b2e63197a1d0f882f1568a80a16d895c8e8d0470c566039742ed92ac339dc5527bd42c50e0fdbfa664e7ff4f9fee5b587ea980ed50
Type spikbladMimetype application/pdf

Authority records BETA

Hussain, Rashida

Search in DiVA

By author/editor
Hussain, Rashida
By organisation
School of Health and Medical Sciences, Örebro University, Sweden
Biomedical Laboratory Science/Technology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 267 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf