Cytokines secreted by splenocytes from mice given KLH, BLG, Ara h1 or Cry1Ab by the i.p. route.

<p>Splenocytes from BALB/cJ mice after i.p. administration of PBS (white bars), 1 µg (gray bars) or 100 µg (black bars) of KLH, BLG, Ara h1 or Cry1Ab were reactivated <i>ex vivo</i> with 50 µg of the corresponding antigen. Expected results were obtained from positive (ConA) and negative controls (medium alone) (data not shown).</p

Metabolic biomarkers identified from discriminant analysis.

<p>Assignment of significant variables explaining the 4 first components displayed by a linear discriminant analysis performed on OSC-PLS–corrected data corresponding to the late urine collections performed on days 27 and 28.</p

Sandwich immunoassay of Cry1Ab.

<p><b>A.</b> Standard curve obtained with purified Cry1Ab protoxin in sandwich immunoassay using mAb#120 passively adsorbed on microplates as capture antibody and AChE-labeled mAb#95 as tracer. Mean +/− SD is represented. Each point is the result of duplicate measurements, except NSB (EIA buffer alone, n = 8). The limit of detection, defined as the lowest concentration of standard Cry1Ab inducing a signal statistically significantly higher than NSB (i.e. mean+3σ<i>_n</i>₋₁) is shown in the insert as a black line. <b>B.</b> Extracts from reference maize powders containing MON810 maize mass fraction level of 0.5% (●), 1% (▼) or 2.5% (◆) (2- to 250-fold dilutions) or standard Cry1Ab (10 ng/mL, 2- to 128-fold dilutions, ○) were assayed using this sandwich immunoassay. All dilution points were assayed in duplicate. Results are expressed as mean of absorbance values (mAU at 414 nm) +/− SD. No signal was detected for non-GM reference maize (not shown).</p

Specific IgG1, IgG2a and IgE antibodies induced after i.p. administration of purified KLH, BLG, Ara h1 or Cry1Ab.

<p>Specific IgG1 (•, left axis), IgG2a (o, left axis) and IgE (*, right axis) antibodies were assayed in sera of individual BALB/cJ mice after two i.p. administrations of 1 (<b>A</b>) or 100 µg (<b>B</b>) of each protein, without adjuvant. Bars represent the mean of 5 mice per group. Nonspecific binding (NSB) was determined using EIA buffer instead of serum. Limit of detection was determined as the mean of NSB +3σ<i>_n</i>₋₁. a p<0.05 when comparing mice receiving protein with mice receiving PBS (not shown), nonparametric test.</p

Linear discriminant analysis performed on OSC-PLS–corrected data corresponding to the late urine collections performed on days 27 and 28.

<p>The four LD are highly significant (p<0.0001) and the 3 respective factorial maps are displayed here: 1×2 (<b>A</b>), 2×3 (<b>B</b>) and 3×4 (<b>C</b>). The cultivar names are displayed on the factorial maps with the .2 and .3 suffixes designating the urine collection performed on days 27 and 28, respectively.</p

A–C: Electrophoretic pattern and IgE-binding analysis of protein extracts from MON810 and non-GM maize:

<p>Protein extracts from MON 810 and its conventional counterpart were separated on 12% acrylamide gel and proteins were stained (<b>A</b>). After transfer to PVDF membrane, IgE binding proteins (i.e. allergens) were revealed by western blotting with the sera of 2 maize-allergic patients, i.e. #20770-MH (<b>B</b>) and #19392-CS (<b>C</b>). Specific IgE concentrations (<b>D</b>) in the same sera were determined on GM (open bar) and non-GM (black bar) protein extract coated plates. Bars represent mean+/−SEM obtained for 3 different dilutions of each serum, each assayed in duplicate.</p

Specific IgE and IgG1 antibodies induced after i.g. or i.p. administration of GM vs non-GM maize extracts.

<p>Mice were given protein extracts from MON 810 or non-GM comparator via the i.p. route (100 µg of protein/mice emulsified with incomplete Freund's adjuvant) on days 1 and 15 (n = 8/group) or via the i.g. route (1 mg of protein mixed with 10 µg of cholera toxin) on days 1, 7, 13, 19 and 25 (n = 10/group). Ten mice received PBS via the i.g. route (PBS mice). Specific IgE (<b>A</b>) and IgG1 (<b>B</b>) antibodies were assayed on microtiter plates coated with non-GM (open circles) or MON810 (black symbols) maize protein extracts. For each group, data of individual serum samples collected on day 30 and the corresponding median values are expressed as absorbance values (mAU at 414 nm). Sample dilutions were 1/40 for assays of IgE antibodies, and 1/4000 (i.g. treated groups) or 1/200000 (i.p. treated groups) for assays of IgG1 antibodies.</p

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 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 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