Sensitivity of Chloride Efflux vs. Transepithelial Measurements in Mixed CF and Normal Airway Epithelial Cell Populations

Illek, Beate; Lei, Dachuan; Fischer, Horst; Gruenert, Dieter C.

doi:10.1159/000324011

Cited by 11 publications

(9 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…aeruginosa binding to and type III secretion of exotoxins into Calu-3 airway epithelial cell monolayers occurs selectively at basolateral membranes and not at apical membranes in cells grown both as islands and on filters [ 18 , 30 ]. (iii) Activation of CFTR by forskolin and genistein in 16HBE41o- cells appears to occur similarly whether cells are grown on filters or on plastic in tissue culture well plates [ 31 ]. (iv) The release of cytosolic amino acids during physical wounding would be expected to occur similarly in polarized and nonpolarized epithelial cells.…”

Section: Discussionmentioning

confidence: 99%

Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

Schwarzer

Fischer²,

Machen

2016

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds.

show abstract

Section: Discussionmentioning

confidence: 99%

Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

Schwarzer

Fischer²,

Machen

2016

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…Ussing chamber studies were used to characterize the electrophysiological properties of epithelial cells, such as relevant ion channel expression and transepithelial resistance. Experiments were performed using a static chamber system (World Precision Instruments, Sarasota FL) . Apical and basolateral compartments were electrically isolated and separated by the polarized epithelial layer.…”

Section: Methodsmentioning

confidence: 99%

Establishment of an immortalized human subglottic epithelial cell line

et al. 2019

View full text Add to dashboard Cite

Objective: Translational research into subglottic disease is restricted by the availability of primary human tissue originating from this subsite. Primary epithelial cells are also limited by their inability to survive beyond several divisions in culture outside of the body. Specific subglottic cell lines, useful for in vitro studies, have not yet been described. We therefore demonstrate what we believe to be the first immortalized subglottic epithelial cell line.Methods: Subglottic tissue was derived from a single adult patient's neoplasia-free human subglottic brushing specimen. Cells were immortalized using a lentiviral vector expressing simian virus 40 T antigen. Karyotyping was performed on the transformed cells using single nucleotide polymorphism array comparative genomic hybridization. Transformed cells were phenotypically characterized by light microscopy, immunohistochemistry, and electrophysiology studies.Results: The immortalized subglottic cell line (SG01) was able to divide successfully beyond 20 passages. Karyotyping demonstrated no significant genomic imbalance after immortalization. The cells demonstrated normal epithelial morphology and cytokeratin expression throughout. SG01 cells were also successfully cultured at air-liquid interface (ALI). At ALI cells demonstrated cilia, mucus production, and relevant ion channel expression.Conclusion: The novel SG01 subglottic epithelial cell line has been established. This cell line provides a unique resource for researchers to investigate subglottic diseases, such as subglottic stenosis.

show abstract

“…Approximately 10 5 cells were plated onto each insert and grown for 9–11 days. Snapwell inserts were mounted via sliders into Easy Mount Ussing chambers (Physiologic Instruments, San Diego, Calif., USA) and short circuit current (I SC ) measurements were performed as previously described for CFBE41o- monolayers (Illek et al 2010). Briefly, a typical four-electrode voltage clamp (VCC MC6, Physiologic Instruments, San Diego, Calif., USA) was connected via Ag/AgCl electrodes (World Precision Instruments, Sarasota, Fla., USA) to the solutions through 3% Agar bridges containing 1 M KCl.…”

Section: Methodsmentioning

confidence: 99%

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

Semir

Maurisse

et al. 2012

Cell Tissue Res

View full text Add to dashboard Cite

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.

show abstract

Sensitivity of Chloride Efflux vs. Transepithelial Measurements in Mixed CF and Normal Airway Epithelial Cell Populations

Abstract: Background/Aims: While the Cl -efflux assays are relatively straightforward,

Cited by 11 publications

References 40 publications

Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

Establishment of an immortalized human subglottic epithelial cell line

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

Contact Info

Product

Resources

About