Development of a new technique for recovery of cytokines from inflammatory sites in situ.
We attempted to recover cytokines from nasal mucosal surface following allergen challenge. Repeated lavage of nasal mucosa of seven allergic patients was done, but we failed to detect IL-1 beta in the lavage samples even in ten-fold concentrated materials. Therefore, we developed a new technique to recover cytokines using filter strips. Small filter strips were placed on nasal turbinates for 10 min at different time points after allergen challenge. The strips were air-dried, and stored. For recovery of cytokines individual strips were washed with small volumes of Hepes buffer containing 0.3% human serum albumin. Eluates were assayed for the presence of IL-1 beta and GM-CSF using commercially available ELISA. We were able to detect IL-1 beta and GM-CSF in eluates. Both cytokines were consistently detectable in the late phase allergic reaction peaking at 5 h. Nasal challenge with saline failed to detect any cytokine during the 7 h observation period. In standardization experiments known quantities of IL-1 beta and GM-CSF were applied to filter strips and the recovery ranged from 67 to 89%. Thus, we developed a simple technique of recovery of cytokines from inflammatory mucosa in situ.
Alam
R
R
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77550.
Sim
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TC
Hilsmeier
K
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Grant
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JA
D000485:Allergens / Q000276:immunology; D004391:Dust; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005374:Filtration; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000302:isolation & purification*; D006801:Humans; D007375:Interleukin-1 / Q000302:isolation & purification*; D009297:Nasal Mucosa / Q000276:immunology*; D009299:Nasal Provocation Tests; D012220:Rhinitis / Q000276:immunology*
D000485:Allergens; D004391:Dust; D007375:Interleukin-1; D016178:Granulocyte-Macrophage Colony-Stimulating Factor