Serological and clinical information for the 34 peanut allergic patients recruited in different European centres, within the Europrevall European project, used in EAST studies, basophil histamine release tests and/or PBMC proliferation tests.

<p>HEP: histamine equivalent prick test; nd: not determined. Where known, other allergies of the subjects are indicated.</p><p>The origin of the sera is as follows: 2A: Zurich, 52A: Amsterdam, 54A-82: Arnhem, 1682-2305: Vienna; 03-0043 till 12-0048 EuroPrevall Serum Bank.</p><p>Grading is based on Brockow and Ring and on Sampson <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023998#pone.0023998-Brockow1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023998#pone.0023998-Sampson1" target="_blank">[42]</a>. Grade 1 = dermal symptoms; grade 2 = gastro-intestinal problems like nausea and or cramping; grade 3 = any of the former grades plus vomiting/diarrhoea and respiratory tract problems like throat pruritis or tightness, grade 4 = any of the former grades and respiratory arrest plus cardiovascular problems like hypotension.</p

Effect of heating on secondary structure and oligomeric state of Ara h 2/6.

<p>Far-UV CD spectra of Ara h 2/6 heated alone (<b>A</b>, H-Ara h 2/6) or in the presence of 100 mM glucose (<b>B</b>, G-Ara h 2/6) before heating (N-Ara h 2/6, —) and for heated-cooled protein after heating to 110°C for 15 min (−−−) and 60 min (---). Inset graphs show change in molar residue ellipticity at 228 nm with heating time. Size exclusion chromatography profiles (<b>C</b>) are shown of the Ara h 2/6 before and after heating for 15 min at 110°C in the presence or absence of glucose. Retention volumes of molecular weight standards (size indicated in kDa) are shown by arrow heads. <b>D</b>. Far-UV CD spectra of Ara h 6 purified from roasted peanut.</p

Effect of thermal treatment on the IgE binding capacity of Ara h 2/6.

<p><b>A.</b> IgE capture inhibition curves obtained for sera #05-0209 with N-Ara h 2/6 (•), H-Ara h 2/6 (⧫) or G-Ara h 2/6 (▴). IgE binding capacities of native and heated processed Ara h 2/6 was assessed by competitive assays in the IgE capture format. Inhibition was performed into microtiter plates coated with anti-IgE, and previously incubated with allergic patient sera at convenient dilution. Competition was then conducted by adding increasing concentrations of competitors at the same time as biotinylated N-Ara h 2/6. Competition obtained with Ara h 2/6 purified from roasted peanut is shown as a clear circle. <b>B</b>. Analysis of IC50 (ng/ml) values obtained using an IgE capture inhibition assay with native and heat processed Ara h 2/6 as competitors using 30 individual sera from peanut-allergic patients. Increase in IC50 value corresponds to a decrease in IgE-binding capacity. R-Ara h 2/6: mix of Ara h 2 and Ara h 6 purified from roasted peanut. Bars indicate a significant difference between the 2 corresponding treatments (<i>P</i><0.05, non-parametric Wilcoxon signed rank test).</p

Effect of thermal treatment on cytokine induction capacity.

<p>Fresh PBMC were left unstimulated (Med) or stimulated with N-, H- or G-Ara h 2/6 and production of IL-5 (<b>A</b>), IL-13 (<b>B</b>), IL-10 (<b>C</b>) and IFN-γ (<b>D</b>) was measured after 7 days of culture. Symbols represent peanut-allergic patients #65 (▪), #66 (♦), #70 (▴), #67 (•) and #73 (×). Horizontal bars represent the mean of the five PA responder subjects (solid) and the mean of 7 NA controls (dotted). Asterisks indicate statistically significant differences between the PA and the NA group and # indicate statistically significant differences between the medium and allergen-stimulated cultures for the PA group (*, #: <i>P</i><0.05). No differences between the medium and allergen-stimulated cultures were observed for the NA group.</p

Effect of thermal treatment on the activation of effector cells induced by Ara h 2/6.

<p>(<b>A</b>) Human stripped basophils were passively sensitized with individual sera (#2305) and then incubated with increasing concentrations of N-Ara h 2/6 (•), H-Ara h 2/6 (⧫), and G-Ara h 2/6 (▴), or whole peanut extract from raw (★) or roasted (○) peanut. Histamine release was assayed in corresponding supernatants. Twenty-three sera out of the 35 available corresponding to all sera from Zurich, Amsterdam, Vienna and EPSB, and sera 54A to 58A from Arnhem were used, giving similar results (not shown). No histamine release was induced by the different allergens when using a serum from patients not sensitized to peanut (data not shown). (<b>B</b>) Humanized RBL-2H3 cells were passively sensitized with sera from peanut-allergic patients (#70) and stimulated with increasing concentrations of native (N; ―•―), heated (H; – –⧫– –) or glycated (G; - - -▴- - -) Ara h 2/6. Error bars represent the SD of triplicate values. No β-hexosaminidase release was induced by the different allergens when using sera from non-allergic patients (data not shown). The table presented within the figure represents the average protein concentrations (ng/ml, n = 6) to obtain 50% of the maximum allergen release of the native allergen (EC50). Means without a common letter differ (P<0.05).</p