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Antigen-driven basophil activation is indicative of early Necator americanus infection in IgE-seronegative patients  Franco H. Falcone, PhD, Gary Telford,

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Presentation on theme: "Antigen-driven basophil activation is indicative of early Necator americanus infection in IgE-seronegative patients  Franco H. Falcone, PhD, Gary Telford,"— Presentation transcript:

1 Antigen-driven basophil activation is indicative of early Necator americanus infection in IgE-seronegative patients  Franco H. Falcone, PhD, Gary Telford, PhD, Doreen Hooi, PhD, Alan P. Brown, PhD, Rita Seabra, PhD, Johanna Feary, BMBS, Andrea Venn, PhD, John Britton, MD, David I. Pritchard, PhD  Journal of Allergy and Clinical Immunology  Volume 124, Issue 6, Pages e7 (December 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Percentage of CD203c/CD63 double-positive (+/+) basophils in whole blood of placebo-treated (n = 14, left) versus N americanus–treated (n = 12, right) subjects n weeks after infection. ∗P ≤ .05 and ∗∗P ≤ 0.01 (Mann-Whitney U test), comparing the placebo-treated versus N americanus–treated groups for each time point. Superimposed on the E/S plot are the egg counts (circles; eggs per gram of stool sample expressed as means ± SDs; n = 9). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 A, N americanus E/S–specific immunoglobulins (AU ± SDs). ∗∗∗P < .001, unpaired t test comparing groups at individual time points. B and C, Western blot of anti–N americanus IgG (Fig 2, B) and IgM (Fig 2, C). Lane 1, Negative control; lanes 2 and 3, N americanus–treated group (week 0 and week 12); lanes 4 and 5, placebo group (week 0 and week 12); lane 6, PNG reference serum.12 Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 RT-PCR analysis of IL4 and IL13 mRNA production in blood of subjects 15, 28, 44, 50, and 34 two years after infection with N americanus. Stimulation with E/S results in upregulation of IL4 and IL13 mRNA 1 hour after stimulation. The values for relative n-fold mRNA increase in E/S-treated versus untreated blood were determined by using SYBR Green quantitative RT-PCR in triplicates with glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) and adenine-phosphoribosyltransferase (APRT) as reference genes, calculated by using the Pfaffl equation16 and the experimentally determined efficiencies for each primer pair. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Gating used to detect whole blood basophil activation
Gating used to detect whole blood basophil activation. A, Side scatter (SS, Pmt1Lin) versus forward scatter (FS, FSLin) of whole blood. B and C, Side-scatter versus forward-scatter dot plots of highly enriched basophils (>90% purity) before (Fig E1, B) and after (Fig E1, C) anti-IgE stimulation. D and F and E and G correspond to unstimulated (Fig E1, B) and stimulated (Fig E1, C) basophils, respectively, and illustrate CD203c and CD63 upregulation on activation. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Examples of dot plots (y-axis, CD203c-PE; x-axis, CD63-FITC) of 100 μL of whole blood stimulated with positive controls (C, anti-IgE; D, fMLP 10 μmol/L), negative controls (A, buffer; B, isotype control), or 10 μg/mL N americanus E/S (E, negative subject; F, positive [infected] subject). Percentages in the upper right quadrant are double-positive CD203c/CD63 basophils based on the initial gating (R1 in Fig E1). The intra-assay (tube–to–tube) variation with this assay was 10% ± 5.99% (mean ± SD, n = 4). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 FACS analysis showing basophil sensitization in 5 infected subjects 2 years after infection. One hundred microliters of whole blood was stimulated with medium alone (control), 10 μmol/L fMLP (positive control), or 10 μg/mL N americanus E/S. Percentages in the upper right quadrant are double-positive CD203c/CD63 basophils based on the initial gating (R1 in Fig E1). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Basophil-enriched PBMCs were stripped with lactic acid to remove surface-bound IgE from the basophils, as described. A shows a forward vs side scatter dot plot of unstripped PBMCs, and the gate used for all other panels. Basophils responded to fMLP (B) or anti-IgE (D) with upregulation of CD203c and CD63 but not to heat-inactivated N americanus E/S (E). Isotype controls (C) show lack of unspecific binding. After IgE stripping, basophils did not respond to anti-IgE stimulation (F), suggesting that surface IgE had been efficiently removed. Incubation with serum 28 (H) but not with the other 4 sera (Fig E4, H; I-K) was able to passively sensitize the donors' basophils. This finding is in agreement with the ELISA data, which did not detect a significant increase of parasite-specific IgE in the sera, with the exception of subject 28's serum. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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