Asthma Clinical Trial
Official title:
Effect of in Vitro Blocking the Common Beta Chain on the Function of Blood, Bone Marrow and Sputum Cells Collected From Asthmatic Donors.
This study examines in vitro blockade of signaling through the β-chain, on viability, activation and differentiation of eosinophils and their progenitors collected in sputum, blood and bone marrow samples pre and post-allergen challenge from mild atopic asthmatic subjects.
The experiments will use sputum samples induced from subjects with mild asthma, undergoing
allergen inhalation challenges. In general, each sample will be composed of >10% neutrophils
and >10% macrophages. Samples collected pre-allergen will have a low frequency of eosinophils
and lymphocytes (<1%), however the percentage of eosinophils will increase to approximately
12% following allergen challenges. The sputum samples will be processed in DPBS (without
dithiothreitol), and the cell suspension will be adjusted to 1 million cells/ml in DMEM with
penicillin and streptomycin. A cytospin will be made for differential cell counts. The mixed
cell population at 5 million cells/ml will be incubated for 48 hours at 37 degrees Celcius ±
β-chain MAb at a concentration of 100 mcg/ml. After 48 hours the cell culture medium will be
removed for assay of cytokines and chemokines by ELISA. Cells will be resuspended in PBS and
Binding Buffer (BD Pharmingen, Cat no. 556454),stained for assessments by flow cytometry, and
analyzed in duplicate.
Experiments will use blood (80 ml) and bone marrow aspirates (5ml) from atopic asthmatics
taken pre and 24hr post allergen challenge. Methylcult micro-culture colonogenic assays will
be performed to enumerate outgrowth of Eo/Baso-CFU and GM-CFU from CD34+ cells populations
collected from the blood and bone marrow samples. Methylcult assays will be performed with
CD34+ enriched cell populations in the presence of IL-5,IL-3 and GM-CSF +/- CSL311. Following
14 days culture, colonies will be enumerated.
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