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

BACKGROUND: 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. DESCRIPTION: 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). RESULTS AND SIGNIFICANCE: 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

Clinical conditions associated with bacterial overgrowth

Emerging evidence suggests that many gastrointestinal diseases are associated-if not caused-by alterations in the gut microbiome. Specific microbiome signatures have been reported for irritable bowel syndrome and inflammatory bowel disease, and these have been associated with pro and antiinflammatory effects contributing to disruption or maintenance of gut homeostasis. Small intestinal bacterial overgrowth (SIBO) is a well-established example of small intestinal dysbiosis. SIBO is defined as a heterogeneous syndrome with excessive and/or abnormal type of bacteria in the proximal small bowel. In addition to increased microbial density, loss of microbial diversity of microbes colonizing the gut mucosa-also labeled as the mucosa associated microbiome (MAM)-is the key finding. However, thus far the translation of this new knowledge into clinical practice and the ability to specifically target alterations of the bacterial colonization of the gut are hampered by the lack of valid and readily available clinical tests to diagnose gut microbial dysbiosis. In the past, culture of jejunal fluid aspirate is considered the “gold standard” for diagnosing dysbiosis, including SIBO, but is rarely used in the clinical setting. Breath tests measuring hydrogen and methane in the exhaled breath-preferentially after a glucose test meal-are simple and widely utilized in the routine clinical setting, but their sensitivity and specificity is far from optimal. In the future utilization of molecular technologies, high throughput will enable us to identify “biomarkers” for various GI and non-GI diseases that will guide therapy