PMC5986952_01
(A) Cytokines and chemokines serum levels during acute exacerbations and clinical remissions in the patient. Healthy donors' values are reported as control. Mean values +- SEM are reported. (B) Cytofluorimetric analysis of cytokines production of peripheral blood T lymphocytes (CD3 gated) before treatment (peripheral blood mononuclear cells were polyclonal stimulated for 6 h in the presence of phorbol myristate acetate plus ionomycin, in the last 4 h brefeldin A was added to inhibit cytokines exocytosis. After stimulation, cells were fixed in formaldehyde, and cell membranes were permeabilized with saponin to permit the staining of intracellular cytokines with the appropriate fluorochrome-conjugated monoclonal antibodies. Samples were evaluated by BLSRDII flow-cytometer (BD-Biosciences, San Jose, CA, USA). At least 104 elements were acquired for each sample and analyzed by FacsDiva BD software. (C) Exacerbation/year and blood eosinophils (absolute value and percentage) before and after the treatment. Mean values +- SEM are reported.
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F1
We observed higher levels of these cytokines during the acute exacerbations than in the clinical remission phases; at remission, values were similar to those observed in healthy controls (n = 5) (Figure 1A). As shown in Figure 1B, the flow-cytometric analysis of intracellular cytokine staining, upon polyclonal stimulation of peripheral lymphocytes collected during an exacerbation phase, showed a Th2-skewed cytokine profile. April 2011 we started intravenous treatment with anti-IL-5 monoclonal antibody mepolizumab (kindly supplied from GlaxoSmithKline) 750 mg every 4 weeks. After a follow-up period of 5 years, a reduction in the rate of exacerbation phases/year (10 +- 1.4 vs 2 +- 0.45; p < 0.005) and in the eosinophils count both in percentage (28.8 +- 3.6 vs 9.8 +- 1; p < 0.001) and absolute value (2,737 +- 5,536 vs 782 +- 104; p < 0.005) were observed (Figure 1C). With regard to CD8+ T lymphocytes, a clear increase of IFN-gamma-producing cells was observed after therapy (Figure S1 in Supplementary Material).