Sezione di Patologia Generale, Dipartimento di Patologia e Diagnostica, University of Verona, Verona, Italy.
PLoS One. 2011;6(7):e22212. doi: 10.1371/journal.pone.0022212. Epub 2011 Jul 21.
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.
评估囊性纤维化跨膜电导调节因子（CFTR）的功能活性，以评估新疗法并定义囊性纤维化（CF）的诊断，这一过程繁琐。已知白细胞表达可检测水平的 CFTR，但尚未在这些细胞中对该分子进行表征。在这项研究中，我们旨在建立和验证一种血液测试，以评估白细胞中 CFTR 的表达和功能。
使用潜在敏感的 DiSBAC(2)(3)探针进行 Western blot、PCR、免疫荧光和单细胞荧光成像分析，以评估 PKA 磷酸化、细胞膜定位的 CFTR 的表达。在非 CF 单核细胞中检测到表达，但在 CF 患者衍生的单核细胞中未检测到或存在截短形式，这些患者携带无义突变。CFTR 激动剂给药可诱导非 CF 供体分离的单核细胞（31 名受试者）发生膜去极化，并且在一定程度上诱导 CFTR 杂合子携带者（HTZ：15 名受试者）发生膜去极化，但在 CF 患者的单核细胞中未能诱导（44 名受试者）。我们提出了一个指数，CF 患者的值明显（p<0.001）低于其他两组。在选定的受试者中测量的鼻电位差与单核细胞测定结果一致（Kappa 统计量为 0.93，95%CI：0.80-1.00）。
CFTR 在人类单核细胞中是可检测的，并且具有功能。我们还表明，可以在 5 毫升外周血中评估 CFTR 相关活性，并设计出一种潜在适用于诊断目的以及基础和转化研究的指数：从药物开发到临床试验中功能结果的评估。
