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Abstract
Techniques are well-established to quantify ciliary beat frequency (CBF), which is often reduced in patients with primary ciliary dyskinesia. This project aims to determine the impact of genetic polymorphisms in the T2R38 (bitter taste) receptor in response to chemical ligands, which is predicted to lead to changes in CBF, which are transient and small in magnitude. These receptors are of interest as they have been shown to ‘sense’ quorum sensing molecules produced by Pseudomonas aeruginosa (Pa) and may thus be disease modifiers in patients with cystic fibrosis. This work describes the development of a rapid CBF assay with sufficient sensitivity to provide a read-out of airway epithelial cell responses to stimulation in vitro.
Air-liquid interface (ALI) cultures were obtained from surplus clinical diagnostic samples. Cells in ALI were exposed to phosphate buffered saline (PBS; negative control) and adenosine 5’-triphosphate (ATP; positive control). Experiments were conducted in temperature-controlled wells under a 40× light microscope. Cilia were imaged by high-speed video camera and CBF expressed as a ratio of stimulated/basal frequencies.
CBF increased in ALI cultures exposed to ATP (n = 4) but not PBS (n = 3), in experiments at 24–25˚C; this effect was not seen at 37 ˚C. Mean ±SEM stimulated/basal CBF ratio was 1.00 ± 0.05 in the PBS-exposed cells, and 1.31 ± 0.08 in the ATP-exposed cells (p = 0.029). Inter-observer variability (n = 2) was lower than within-sample CBF variability (95% limits of agreement from -0.66 to 1.62 Hz). Intra-observer variability was good with 95% limits of agreement between -0.31 to 0.52 Hz.
An assay has been developed to detect rapid changes in CBF in ALI cultures using ATP as a positive control. Further work is being undertaken to a) optimise this assay in epithelial cells in suspension, thus increasing throughput, and b) assess more relevant chemicals and culture media. Once optimised, this assay will be used to study the effects of Pa quorum sensing molecules on ciliated epithelial cells in vitro, from patients of varying TAS2R38 genotypes.
