Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. I...

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Veröffentlicht in:PloS one Jg. 6; H. 7; S. e22212
Hauptverfasser: Sorio, Claudio, Buffelli, Mario, Angiari, Chiara, Ettorre, Michele, Johansson, Jan, Vezzalini, Marzia, Viviani, Laura, Ricciardi, Mario, Verzè, Genny, Assael, Baroukh Maurice, Melotti, Paola
Format: Journal Article
Sprache:Englisch
Veröffentlicht: United States Public Library of Science 21.07.2011
Public Library of Science (PLoS)
Schlagworte:
Adolescent
Adult
Aged
Algorithms
Amiloride - pharmacology
Biology
Blood
Blood tests
Cell Line
Child
Child, Preschool
Chloride
Clinical trials
Conductance
Cystic fibrosis
Cystic Fibrosis - blood
Cystic Fibrosis - diagnosis
Cystic Fibrosis - physiopathology
Cystic Fibrosis Transmembrane Conductance Regulator - blood
Cystic Fibrosis Transmembrane Conductance Regulator - metabolism
Depolarization
Diagnosis
Diagnostic systems
Drug development
Epithelial Cells - drug effects
Epithelial Cells - metabolism
Evaluation
Female
Fibrosis
Fluorescence
Genetic counseling
Genotype & phenotype
Hematologic Tests - methods
Heterozygote
Humans
Immunofluorescence
Indexing
Infant
Kinases
Leukocytes
Male
Medical research
Medicine
Membrane potential
Membrane Potentials - drug effects
Middle Aged
Monocytes
Monocytes - drug effects
Monocytes - metabolism
Mutation
Older people
Patients
Peripheral blood
Phenotype
Physiology
Protein kinase A
Pseudomonas aeruginosa
Resistance
Single-Cell Analysis
White blood cells
Young Adult
Italy
ISSN:1932-6203, 1932-6203
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Zusammenfassung:Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials.
Bibliographie:ObjectType-Article-1
SourceType-Scholarly Journals-1
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Conceived and designed the experiments: CS PM. Performed the experiments: CA JJ ME GV MV MR. Analyzed the data: CA JJ LV ME MB. Contributed reagents/materials/analysis tools: BMA PM LV MB. Wrote the paper: CS PM.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0022212