Effect of in Vitro Blocking the Common Beta Chain on Cell Viability in Asthma
Effect of in Vitro Blocking the Common Beta Chain on Cell Viability in Asthma
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.
Inclusion Criteria:

 -  Male and female volunteers 18 through 65 years of age.

 -  General good health

 -  Mild to moderate, stable, allergic asthma

 -  History of episodic wheeze and shortness of breath; FEV1 at baseline at least 70% of
 the predicted value

 -  Able to understand and give written informed consent and has signed a written informed
 consent form approved by the investigator's REB

 -  Positive methacholine challenge

 -  Positive skin-prick test to common aeroallergens (including cat, dust mite, grass,
 pollen)

 -  Positive allergen-induced airway bronchoconstriction (a fall in FEV1 of at least 20%
 from baseline)

 Exclusion Criteria:

 -  A worsening of asthma or a respiratory tract infection within 6 weeks preceding study
 entry

 -  Use of corticosteroids, immunosuppressives, anticoagulants (warfarin or heparin)
 within 28 days prior to randomization into the study

 -  Use of nonsteroidal anti-inflammatory drugs (NSAIDs) within 48 hours of dosing or
 aspirin with 7 days of dosing

 -  Have chronic use of any other medication for treatment of allergic lung disease other
 than short- and intermediate-acting ß2-agonists or ipratropium bromide

 -  Use of caffeine-containing products or medications for 12 hours or alcohol or over the
 counter drugs including aspirin, cold and allergy medications for 48 hours or inhaled
 bronchodilators for 8 hours prior to methacholine and allergen challenges

 -  Use of tobacco products of any kind currently or within the previous 12 months, or
 smoking history > 10 pack years.

 -  Lung disease other than mild to moderate allergic asthma

 -  Unwillingness or inability to comply with the study protocol for any other reason.