Abstract
BackgroundEvaluation 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.DescriptionWestern blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC2(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).Results and SignificanceCFTR 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.
Highlights
IntroductionCystic fibrosis (CF) is primarily a disorder of electrolyte transport by epithelial cells in which an anion channel, activated by cyclic adenosine monophosphate (cAMP)-dependent kinase (the cystic fibrosis transmembrane conductance regulator-CFTR) is defective and represents the most common autosomal recessive disease with lethal consequences in Caucasians
Cystic fibrosis (CF) is primarily a disorder of electrolyte transport by epithelial cells in which an anion channel, activated by cyclic adenosine monophosphate-dependent kinase is defective and represents the most common autosomal recessive disease with lethal consequences in Caucasians
CFTR expression in epithelial cells and monocytes Western blotting analysis of cell lysates from pancreatic and bronchial epithelial cell lines expressing wild-type CFTR (Suit-2 and 16HBE14o-) allows to identify the presence of all the major CFTR bands described in the literature, namely bands A, B and C (Figure 1A)
Summary
Cystic fibrosis (CF) is primarily a disorder of electrolyte transport by epithelial cells in which an anion channel, activated by cyclic adenosine monophosphate (cAMP)-dependent kinase (the cystic fibrosis transmembrane conductance regulator-CFTR) is defective and represents the most common autosomal recessive disease with lethal consequences in Caucasians. Functional CFTR is currently evaluated in humans using ex vivo and in vivo assays, the former method utilizing rectal biopsies [4]. This approach permits the direct recording of transepithelial currents (Intestinal Current Measurements, ICM) but requires an excision of a tissue sample. Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. Expression of PKA phosphorylated, cell membranelocalized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80–1.00)
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