Respiratory (Asthma & COPD)

 

Overview

Asthma is one of the most common chronic respiratory diseases in developed countries. For the majority of asthmatics their symptoms are satisfactorily controlled by the regular use of inhaled glucocorticoids (GC). Eosinophils are key pro-inflammatory cells in the asthmatic lung where their cytotoxic products cause damage to the airway epithelium, tissue inflammation and airflow obstruction. Eosinophil adhesion to and transmigration through the endothelial cells lining the post-capillary venules are key events in their accumulation in the asthmatic lung. Montelukast (MLK) is part of a new class of anti-asthma drugs that are antagonists to cysLT1R reducing eosinophil migration. Evidence is accumulating that MLK may have additional anti-inflammatory effects on eosinophil function which were further investigated in this study. These experimental conditions can easily be studied using Cellix's microfluidic pumps and biochips. The dimensions of the biochips can mimic those of human capillaries thereby enabling researchers to simulate in vivo microenvironments. Cellix's 8-channel Mirus Evo Nanopump enables researchers to conduct up to 8 assays simultaneously in microfluidic biochips while single assays may be conducted using the ExiGo pump.

 

Assay Examples:

Eosinophil - Adhesion Assay - Asthma

Eosinophil – Inflammation – Adhesion Assay

 

Eosinophil - Adhesion Assay - Asthma

Aim: To determine novel anti-inflammatory effects of MLK on resting and GM-CSF-stimulated eosinophils using the Cellix VenaFlux™ platform to mimic physiological adhesion to rhVCAM-1
Vena 8™ biochip micro-channels were coated for 1 hour in humid conditions at ambient temperature with either rhVCAM-1 or BSA (both 10 μg mL-1 in HBSS containing Ca2+ and Mg2+). All capillaries were then coated with BSA to occupy non-specific binding sites.
Eosinophils were infused into the capillaries via Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays at stepwise increase in shear stress, from 0 to 5 dyne cm-2, 1 minute per shear stress level. For experiments with GM-CSF-stimulated (1 ngmL-1) eosinophils, the
cytokine was added to the warmed cells and incubated at 37°C for 20 mins prior to commencing the flow assay.
Results: MLK (10 nM and 100 nM) gave partial (~40%) but significant (P<0.05) inhibition of unstimulated eosinophil adhesion torhVCAM-1 at 2 dyne cm-2. GM-CSFstimulated eosinophil adhesion under flow was characterised by greater cell flattening with significant (P<0.05) inhibition of adherent cell numbers by 100 nM MLK observed. This effect appeared specific for MLK as the analogue MK571 had no significant effect on eosinophil adhesion to VCAM-1. LTC4 released from unstimulated or GM-CSF-treated eosinophils did not contribute to their adhesion to VCAM-1 as the leukotriene biosynthesis inhibitor MK886 had no inhibitory effect while exogenously added LTC4 did not enhance eosinophil adhesion. In contrast,LTD4, enhanced eosinophil adhesion to VCAM-1; an effect blocked by MLK.

 

 

Eosinophil – Inflammation – Adhesion Assay

Aim: To determine the effect of concentration of Levocetirizine on GM-CSF stimulated eosinophil adhesion under flow conditions to rhVCAM-1 using the Cellix VenaFlux™ platform.

Vena 8™ biochip micro-channels were coated for 1 hour in humid conditions at ambient temperature with either rhVCAM-1 or BSA (both 10 μg mL-1 in HBSS containing Ca2+ and Mg2+). All capillaries were then coated with BSA to occupy non-specific binding sites.
Eosinophils were infused into the capillaries via Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays at stepwise increase in shear stress, from 0 to 5 dyne cm-2, 1 minute per shear stress level. For experiments with GM-CSF-stimulated (1 ngmL-1) eosinophils, the
cytokine was added to the warmed cells at the same time as levocetirizine and incubated at 37°C for 20 mins prior to commencing the flow assay.
Results: Pre-incubation of eosinophils with GM-CSF increased the adhesion level by more than 50%, and also rapidly generated significantly more flattened cells at both the 5 and 15 min time-points. Levocetirizine significantly inhibited GM-CSF stimulated eosinophil adhesion to VCAM-1 and the ability of eosinophils to flatten on rhVCAM-1, in a dose-dependent manner, at both 5 and 15 min. Optimal inhibition of adhesion was observed at a levocetirizine concentration of 10-8 M with an EC50 of 10-9 M.