Objective: Evaluation of the functional cystic fibrosis transmembrane conductance regulator (CFTR) to assess new therapies and define diagnosis of atypical cases of cystic fibrosis (CF) is cumbersome and practicable only for selected subjects in few centers. It is known that leukocytes express detectable levels of CFTR. Our goal was to develop methods suitable to assess CFTR expression and activity in leukocytes obtained from healthy subjects or patients with cystic fibrosis ex vivo. Methods: Western blotting and flow cytometry, and cell membrane depolarization, by single-cell fluorescence imaging, using the potential-sensitive probe bis- (1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2), were used. Results: We detected expression of a CFTR isoform in monocytes whose specificity is supported by the lack of expression in cells derived from a patient homozygous for nonsense mutations. The isoform detected in monocytes is different from the pattern observed in epithelial bronchial and pancreatic cells suggesting a different post-translational processing. Functional analysis has shown that, upon stimulation, only non-CF donors and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ) monocytes showed an increase in the fluorescent signal, while monocytes from CF patients failed to respond and presented a spontaneous depolarization signal higher than controls and HTZ monocytes. This feature allows CF patients to be distinguished from all other individuals with high sensitivity and specificity (>95%). As a reference assay, Nasal Potential Difference was measured in selected subjects in parallel to the monocytes assay. Conclusions: These findings suggest that evaluation of CFTR expression by Western blotting and flow cytometry and the measurement of its activity in monocytes by optical techniques may represent a surrogate biomarker suitable for assessment of CFTR expression and function in both basic and translational research, including drug development and diagnostic applications.
New potential biomarkers for the management of cystic fibrosis: measurements of CFTR expression & function in human leukocytes.
SORIO, Claudio;BUFFELLI, Mario Rosario;ANGIARI, Chiara;ETTORRE, Michele;JOHANSSON, Jan Evert;VEZZALINI, Marzia;VERZE', Genny;MELOTTI, Paola Maria
2010-01-01
Abstract
Objective: Evaluation of the functional cystic fibrosis transmembrane conductance regulator (CFTR) to assess new therapies and define diagnosis of atypical cases of cystic fibrosis (CF) is cumbersome and practicable only for selected subjects in few centers. It is known that leukocytes express detectable levels of CFTR. Our goal was to develop methods suitable to assess CFTR expression and activity in leukocytes obtained from healthy subjects or patients with cystic fibrosis ex vivo. Methods: Western blotting and flow cytometry, and cell membrane depolarization, by single-cell fluorescence imaging, using the potential-sensitive probe bis- (1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2), were used. Results: We detected expression of a CFTR isoform in monocytes whose specificity is supported by the lack of expression in cells derived from a patient homozygous for nonsense mutations. The isoform detected in monocytes is different from the pattern observed in epithelial bronchial and pancreatic cells suggesting a different post-translational processing. Functional analysis has shown that, upon stimulation, only non-CF donors and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ) monocytes showed an increase in the fluorescent signal, while monocytes from CF patients failed to respond and presented a spontaneous depolarization signal higher than controls and HTZ monocytes. This feature allows CF patients to be distinguished from all other individuals with high sensitivity and specificity (>95%). As a reference assay, Nasal Potential Difference was measured in selected subjects in parallel to the monocytes assay. Conclusions: These findings suggest that evaluation of CFTR expression by Western blotting and flow cytometry and the measurement of its activity in monocytes by optical techniques may represent a surrogate biomarker suitable for assessment of CFTR expression and function in both basic and translational research, including drug development and diagnostic applications.
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