title: A whole blood assay as a simple, broad assessment of cytokines and chemokines to evaluate human immune responses to Mycobacterium tuberculosis antigens.
abstract: In vitro stimulation of whole blood or isolated peripheral blood cells with specific antigens is used for several purposes. We sought to identify a reliable, reproducible, fast and feasible in vitro method to assess human cellular immune responses to Mycobacterium tuberculosis. In contrast to peripheral blood mononuclear cell (PBMC) culture, a whole blood assay (WBA) provides a more physiological environment, which may provide a broader assessment of serum biomarker, biosignature profiles. Twenty-three asymptomatic individuals with M. tuberculosis infection were recruited. Total cells from the WBA (diluted 1:3 in completed RPMI) and PBMC (2×10(5)cells/ml) plus M. tuberculosis Ag85A, Ag85B, ESAT-6 and Mycobacterium bovis 65kDa were characterized by flow cytometry, then added in 96-well plates and on day 5 plasma and supernatants were harvested for detection of 17 cytokines by a Luminex array system. There was agreement between PBMC and WBA for IL-2, IL-5, IL-6, IL-7, IL-13, IFN-γ, TNF-α, MCP-1 and MIP-1β. There was evidence toward higher IL-10 (p≤0.049) and G-CSF (p≤0.012) plasma production, and higher IL-1β (p≤0.048), IL-4 (p≤0.044), IL-12p70 (p≤0.006), IL-17 (p≤0.002) and GM-CSF (p≤0.049) production for PBMC vs. WBA. Both methods provided virtually no reaction to the internal, negative control. Due to technical issues linked to data out of range, IL-8 data were not considered. These results suggest that, depending on the method employed, PBMC and/or WBA techniques provide fine conditions for the model proposed and thus whole blood cultures are well-suited low-cost proxy-measures during search for serum biomarkers.
authors.affiliation: Laboratório de Imunologia Clínica, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brazil.
authors.forename: Dolores
authors.initials: D
authors.lastname: Silva
authors.forename: Carlos G G
authors.initials: CG
authors.lastname: Ponte
authors.forename: Mariana A
authors.initials: MA
authors.lastname: Hacker
authors.forename: Paulo R Z
authors.initials: PR
authors.lastname: Antas
mesh_terms: D000942:Antigens, Bacterial / Q000276:immunology*; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D009163:Mycobacterium bovis; D009169:Mycobacterium tuberculosis / Q000276:immunology* / Q000378:metabolism; D014397:Tuberculosis, Pulmonary / Q000097:blood* / Q000175:diagnosis
chemical_list: D000942:Antigens, Bacterial; D018925:Chemokines; D016207:Cytokines