Detection of Api m 12 and Ves v 6 in <i>A. mellifera</i> and <i>V. vulgaris</i> venom.

<p>A SDS-PAGE and Coomassie blue staining of the high molecular weight fraction of honeybee venom. The arrow indicates the 200 kDa band that was subjected to MS/MS-based sequencing. B IgE Immunoreactivity of pooled sera from YJV-sensitized patients with the venom of <i>V. vulgaris</i> in Western Blot (AlaBlot™). The arrow indicates the reactive 200 kDa protein band.</p

Domain architecture of Api m 12, Ves v 6 and other vitellogenins.

<p>Comparison of the domain architecture of Api m 12 and Ves v 6 with that of the vitellogenins from the hymenoptera species <i>Bombus ignitus</i> (Genbank accession ACM46019) and <i>Nasonia vitripennis</i> (Genbank accession XP_001607388) as well as with that of vitellogenin allergens of the mites <i>Dermatophagoides pteronyssinus</i> (Der p 14, Genbank accession AAM21322) and <i>Euroglyphus maynei</i> (Eur m 14, Genbank accession AAF14270), the fish <i>Oncorhynchus mykiss</i> (Onc m Vg, Genbank accession CAA63421), and <i>Gallus gallus</i> (Gal d 6, Genbank accession AAA49139). DUF, domain of unknown function; VWD, von Willebrand factor type D domain.</p

Recombinant expression and immunoreactivity of Api m 12 and Ves v 6.

<p>A, B SDS-PAGE and immunoblot analyses of Api m 12 and Ves v 6 recombinantly produced in Sf9 insect cells visualized by Coomassie blue staining, monoclonal anti-V5 epitope antibody and <i>Galanthus nivalis</i> agglutinin (GNA), recognizing terminal mannose 1,2-, 1,3-, and 1,6-linked to mannose. C, D Immunoreactivity of recombinant Api m 12 and Ves v 6 in ELISA using the monoclonal anti-V5 epitope antibody and polyclonal anti-HRP antiserum specific for α1,3-fucose residues, the underlying principle of hymenoptera venom cross-reactive carbohydrate determinant (CCD) reactivity.</p

Effect of TOFA on the differentiation of human FOXP3⁺CD4⁺ T cells.

<p>Naive CD4⁺ T cells of human blood donors (n = 3) were stimulated with anti-CD3/anti-CD28, IL-2 and TGF-β1 to induce FOXP3 expression. In addition to this polarization control, TOFA, Rapamycin or Cycloysporine A were added to the medium in different concentrations. (A) Flow cytometry plots of anti-CD4 and anti-FOXP3 staining are shown for one representative donor. (B) The percentages of FOXP3⁺ cells within the living CD4⁺ T cell population under control, TOFA, Rapamycin and Cyclosporine A condition is given for the three donors. Bars indicate the median.</p

Analysis of murine plasma samples (Non-allergic, Allergic, Allergic-AIT, Allergic-AIT+TOFA) after OVA-aerosol challenge.

<p>(A) Results of the immunoglobulin measurement for OVA-specific IgE, total IgE and total IgG1, and (B) results of the cytokine measurement for IL-6, IL-1β, IL-2 and IL-17A. Bars indicate the median. Gaussian and non-Gaussian distributed results were analyzed by unpaired t test or Mann Whitney test, respectively.</p

Analysis of the cellular BAL fluid compartment and lung histology of mice (Non-allergic, Allergic, Allergic-AIT, Allergic-AIT+TOFA) after OVA-aerosol challenge.

<p>(A) Counts of total BAL cells and total cell count of eosinophils, macrophages, neutrophils and lymphocytes. (B) Relative percentages of eosinophils, macrophages, neutrophils and lymphocytes within the total BAL cells. Shown is the median with range. Gaussian and non-Gaussian distributed results were analyzed by unpaired t test or Mann Whitney test, respectively. (C-F): Lung histology (H&E staining). (C) Non-allergic. (D) Allergic. (E) Allergic-AIT. (F) Allergic-AIT + TOFA. Arrows indicate inflammatory infiltrates; scale bar: 50 μm.</p

Analysis of the soluble BAL fluid compartment (Non-allergic, Allergic, Allergic-AIT, Allergic-AIT+TOFA) after OVA-aerosol challenge.

<p>Results of the cytokine measurement for IL-4, IL-13, IL-5, IL-17A, IL-6, TNF-α, IL-2, IL-1β, IL-10, IL-12p70, IFN-γ and CXCL1. Bars indicate the median. Gaussian and non-Gaussian distributed results were analyzed by unpaired t test or Mann Whitney test, respectively.</p

Schematic overview about the different <i>in vivo</i> treatment schemes (sensitization, AIT, TOFA administration, challenge).

<p>The treatment of four experimental groups of mice (n = 9–14) is shown. AIT, allergen-specific immunotherapy; i.p, intraperitoneal; s.c., subcutaneous.</p

Criteria for selection of mutant mouse line.

<p>^apublished data</p><p>^boriginal data from GMC screen</p><p>Criteria for selection of mutant mouse line.</p

Lung transcriptome comparison of mutant versus wild type animals following OVA challenge.

<p>(A) Summarized heat map of genes regulated after OVA challenge in all three mutant mouse lines compared to the respective challenged control littermates. Genes with similar expression patterns are grouped together (A to G). Color code indicates the mean fold changes of the respective genes in one group for each OVA challenged mutant mouse compared to the mean of the corresponding challenged wild type littermates. Orange represents up- and blue down-regulation in the mutant mice. The column “# genes” displays the number of genes that are included in each gene group A to G (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134503#pone.0134503.s003" target="_blank">S2D Fig</a> shows the corresponding data for individual genes). (B) Venn diagram representing the overlap of gene regulation. (C) Summarized heat map of overlapping gene expression in at least 2 of the 3 mutant mouse lines. Grey boxes indicate that there is no significant regulation of these genes in the respective mutant mouse lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134503#pone.0134503.s003" target="_blank">S2E Fig</a> shows the data for individual genes of this panel).</p