Regular albuterol or nedocromil sodium — effects on airway subepithelial tenascin in asthma

AbstractBoth albuterol and nedocromil sodium have been recognized to possess certain anti-inflammatory properties. However, there are no data on the impact of these drugs on the pathophysiology of the bronchial extracellular matrix in asthma characterized by enhanced tenascin (Tn) expression, known to occur proportional to the severity of asthma. This paper reports data from a morphometric study on the effects of regular treatment with inhaled albuterol or nedocromil sodium on the extent of bronchial subepithelial deposition of Tn, collagen types III, IV, and VII and mucosal infiltration with macrophages.Thirty-two patients (14 women) with chronic asthma, aged 38·7 years (median) with a median forced expiratory volume in 1 sec (FEV1) of 74·4% predicted, were selected to undergo fibre-optic bronchoscopy with bronchial biopsies before and after 12 weeks of treatment with either inhaled albuterol 0·2 mg or nedocromil sodium 4 mg four times daily according to a double-blind protocol. Cryostat sections of the biopsy specimens were studied by indirect immunostaining techniques using monoclonal antibodies and computer-assisted quantitative image analysis.Albuterol treatment significantly reduced the median thickness of subepithelial Tn expression from 9·7 to 6·3 μm (P=0·023) and macrophage numbers in the epithelium (P=0·034), lamina propria (P=0·039) and entire mucosa (P=0·033), whereas nedocromil sodium had no effect. Expression of the collagen types was not affected by either treatment. There was no identifiable statistical difference between the two treatments for any of the outcome variables measured. Nevertheless, the results demonstrate that even a short-acting β2-agonist may exert anti-inflammatory potential sufficient to interfere with the basic mechanisms of asthma as shown by reduction of subepithelial Tn content and mucosal macrophage count

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z’ value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3R cells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies