CB-derived CD4⁺CD25⁺ T cells are deficient with regard to their regulatory function harboring a subset with limited net suppressive effects.

<p>(<b>A</b>) Specificity of proliferation to BLG and BLG+LPS is displayed via CFSE labeling experiments. (<b>B</b>) Inhibition experiments were performed using and increasing amount of MACS-sorted CD4⁺CD25⁺ T cells to the CD4⁺CD25⁻ fraction in presence of irradiated APCs. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ cells. Graphs indicate the means of 5–8 independent experiments with means +/− SEM. Wilcoxon sign rank test was applied. P-values of less than 0.05 were considered significant. (<b>C</b>) CBMCs were separated via FACS on day 0 according to their CD25 and CD127 expression. Four subgroups (CD4⁺CD25^highCD127^low (F1), CD4⁺CD25^intermediateCD127^low (F2), CD4⁺CD25^intermediateCD127^high (F3), and CD4⁺CD25⁻ T cells (CD25⁻)) were obtained. An inhibition assay was performed with the first three fractions using the CD4⁺CD25⁻ T cells as effector cells. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ subgroup. Graphs indicate the means of 4 independent experiments and SEM. (<b>D</b>) Real-time PCR analysis was performed to analyze the FOXP3, TGF-β, and IL-10 expression for further examination of the four fractions. Data represents three independent experiments showing the normalized expression of means +/− SEM.</p

Upon priming with BLG, cord blood derived CD4⁺CD25⁺ T cells become highly suppressive.

<p>(<b>A</b>) Cord blood-derived mononuclear cells were co-cultured for 6 days with the antigen BLG. At day six CD4⁺CD25⁺ T cells were MACS-sorted and an inhibition assay was performed with the CD4⁺CD25⁻ (frozen on day 0, without prior antigen exposure) and stimulated with α-CD3 or BLG. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ cells. Results are representative for 6–9 independent experiments. Wilcoxon signed rank test was applied. P-values of less than 0.05 were considered significant (*); p<0.005 (**); p<0.001 (***). (<b>B</b>) Real-time PCR analysis was performed to quantify the gene expression of Treg-related markers (FOXP3, GARP, RUNX 1, and RUNX 3) after allergen exposure. Results are representative for three independent experiments showing the normalized expression. CD4⁺CD25⁻ T cells are displayed with white and CD4⁺CD25⁺ T cells with black squares.</p

Comparable Treg-related transcription factor and marker expression in CB and PB on day 0.

<p>(<b>A</b>) Cord and peripheral blood derived mononuclear cells were analyzed via flow cytometry on day 0 without prior stimulation. Surface and intracellular staining was performed and one out of three representative experiments is shown. (<b>B</b>) Real-time PCR analysis was performed to quantify the gene expression of Treg-related markers (FOXP3, GARP, TGF-β, RUNX 1, RUNX 3, and IL-10). Results are representative for three independent experiments showing the normalized expression. CD4⁺CD25⁻ T cells are displayed with white and CD4⁺CD25⁺ T cells with black squares.</p

Immunohistochemistry with FcεRIα specific antibody (mAb 15-1) on snap-frozen intestinal specimens from (A) the esophagus, (B) the stomach, (C) the duodenum, and (D) the colon.

<p>FcεRIα-positive cells (red) are frequently found in the esophagus, the stomach, and the duodenum (black arrows). (E) shows isotype control with mouse IgG1. Goblet cells in the duodenum and the colon revealed non-specific binding of antibodies. Original magnification x20. Bottom row (F-J) shows details from A-E. Representative specimens from n = 10.</p

Patients’ characteristics.

<p>IBD: inflammatory bowel disease.</p

FcεRI mRNA expression levels in the upper gastrointestinal tract under inflammatory conditions.

<p>(A) FcεRIα-, (B) FcεRIβ-, and (C) FcεRIγ-mRNA expression levels in specimens from children with gastritis/esophagitis (open squares), celiac disease (open triangles), inflammatory bowel disease (IBD) (open diamonds), and normal mucosa (open circles). * p<0.05, Kruskal-Wallis test.</p

c-kit positive mast cells in the esophagus epithelium express FcεRIα.

<p>(A) FcεRIα is visualized with mAb Cra1 (green, first panel). Mast cells are shown with c-kit as a marker (red, second panel). Cell nuclei are visualized with DAPI staining (blue). (B) shows higher magnifications from (A). FcεRIα is expressed on esophageal mast cells (B, white arrows). Representative specimens from n = 3.</p

Quantitative RT-PCR for the three subunits of the high affinity IgE-receptor was performed on intestinal biopsies from pediatric patients.

<p>(A) FcεRIα mRNA transcripts were found in all esophageal biopsies and with varying frequency in more distal biopsies (left panel). Similarly, the highest frequency of FcεRIβ mRNA-positive specimens was found in the esophagus (middle panel). The common Fc-γ chain was detected in the majority of specimens from the entire GI tract (right panel). Black stacked-bars represent target-positive specimens, and white stacked-bars represent target-negative specimens. (B) The highest levels of FcεRIα mRNA transcripts were found in the esophageal mucosa (left panel), while FcεRIβ mRNA expression peaked in the gastric mucosa (middle panel). Esophageal specimens revealed the lowest FcεRIγ mRNA expression compared to the stomach, terminal ileum, colon, and rectum (right panel). * p<0.05, Mann-Whitney-U test.</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