Effect of in Vitro Blocking the Common Beta Chain on Cell Viability in Asthma
|Study Design:||Observational Model: Cohort
Time Perspective: Prospective
|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.|
- Sputum Collection [ Time Frame: 7 hours post allergen ]Three baseline sample collections will ensure sufficient numbers of cells are collected for proposed outcomes. Post allergen cells will be examined at time points when these populations are known to be at their highest frequency.
Biospecimen Retention: Samples Without DNA
|Study Start Date:||November 2012|
|Study Completion Date:||September 2014|
|Primary Completion Date:||September 2014 (Final data collection date for primary outcome measure)|
Determination of the effects of a β-chain monoclonal antibody (MAb) on the function of cells naturally activated by in vivo allergen exposure, from donors with allergen-induced asthma
Allergen extract is administered by inhalation.
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.
Please refer to this study by its ClinicalTrials.gov identifier: NCT01759849
|McMaster University Medical Center|
|Hamilton, Ontario, Canada, L8N 3Z5|
|Principal Investigator:||Gail M Gauvreau, PhD||Hamilton Health Sciences Corporation|