Nasal Cytokines (PCS1, PCS2, PMBC, PCS3)

Sample Collection

See Collection and Handling of Nasal Lavage Samples for details on sample collection and preparation.

Assay Procedures (PCS1¹, PCS2, PCS3)

Nasal wash fluid was assayed (in duplicate) for interleukin (IL)-1β, IL-5 (PCS3 only), IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-α, and IFN-γ (PCS2 only) using commercially available enzyme-linked immunosorbent assays (ELISAs; Endogen).  Levels were converted to concentrations by correcting for dilution. There, the dilution of nasal secretion in the aspirate/wash is estimated by measurement of the urea concentration using a coupled enzyme reaction involving urease and glutamate dehydrogenase (Sigma Diagnostics Kit No. 66-UV, Sigma Chemical Co.).  Briefly, 20 µl of aspirate/wash is added to 300 µl BUN (Endpoint) reagent at room temperature and the absorbance after five minutes is measured at 340 nm on a spectrophotometer.  Validity of each run is assessed by inclusion of a diluent blank and a urea standard (Sigma).  The dilution factor in an aspirate/wash is calculated by dividing the urea concentration in mg/dl into the assumed normal blood urea concentration of 10 mg/dl.

Nasal cytokine assays for PCS1, PCS2, and PCS3 were performed at the ENT Research Laboratory of the Children’s Hospital of Pittsburgh.

Assay Procedures (PMBC)

Nasal wash fluid was assayed for interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ using the BioSource Ten-plex Bead Immunoassay and methods provided by the manufacturer (BioSource International, Camarillo, CA).  Because the Ten-plex allows simultaneous measurement of multiple biological markers in a single sample, all cytokines were assayed at once.  Antibodies to IL-1β, IL-6, TNF-α, and IFN-γ were covalently linked to beads (microspheres) with a unique fluorophore. Samples were incubated with antibody-coated beads in a 96-well filter plate, washed, and then incubated with a biotinylated antibody specific for a different binding site on the same analyte.  After washing to remove excess biotinylated antibody, samples were incubated with streptavidin-RPE (fluorescent), which binds to biotinylated antibody for detection.  Samples were washed to remove unbound dye, and bead-bound fluorescence was quantified using the Bio-Plex system (Bio-Rad Laboratories, Inc., Hercules, CA). The intensity of fluorescence was measured and converted to the concentration of each cytokine.

Cytokine assays were performed at the Immunologic Monitoring and Cellular Products Laboratory (IMCPL), University of Pittsburgh Cancer Institute (http://www.upci.upmc.edu/iml/; http://www.upci.upmc.edu/cpl/). The IMCPL has an established quality assurance/quality control program for cytokine determinations and a long-standing expertise in cytokine measurements for both clinical and research programs.

¹ In PCS1, nasal secretions were assayed only for IL-8.

Reference

Janicki-Deverts, D., Cohen, S., Doyle, W. J. Turner, R. B., & Treanor, J. J.  (2007). Infection-induced pro-inflammatory cytokines are associated with decreases in positive affect, but not increases in negative affect.  Brain, Behavior and Immunity, 21, 301-307.