Level of Foxp3 expression.

<p>The specific mRNAs in OVA-stimulated mice splenocytes (incubated with OVA for 72hrs) were quantified by qRT-PCR. The results are presented as mean mRNA expression (mean±SE, n = 8 for each). The relative levels of Foxp3 expression were calculated by referring to the HPRT (hypoxanthine guanine phosphoribosyltransferase) in each sample. AD without ASIT: OVA-sensitized untreated mice; ASIT with OVA: OVA-sensitized mice treated by ASIT with OVA; ASIT with sOVA: OVA-sensitized mice treated by ASIT with sOVA; placebo: PBS-sensitized mice. *, p<0.05.</p

Levels of cytokines.

<p>The levels of cytokines in supernatants of OVA-stimulated mice splenocytes (incubated with OVA for 72hrs) were quantified by ELISA. The results are presented as mean IL concentration (mean±SE, n = 8 for each). AD without ASIT: OVA-sensitized untreated mice; ASIT with OVA: OVA-sensitized mice treated by ASIT with OVA; ASIT with sOVA: OVA-sensitized mice treated by ASIT with sOVA; placebo: PBS-sensitized mice. Level of (a) the IL-4, (b) IL-5, (c) IL-12, (d) IFN-γ, and (e) IL-17; (f) IL-4/IFN-γ ratio in mice with ASIT. *, p<0.05 versus “AD without ASIT”; **, p<0.05 versus “placebo”; #, p<0.05 versus “ASIT with OVA”.</p

Analysis of OVA-specific IgG.

<p>The levels of antibodies in sera obtained before, during, and after ASIT were detected and quantified by ELISA. The results are presented as mean antibodies concentrations (mean±SE, n = 8 for each). AD without ASIT: OVA-sensitized untreated mice; ASIT with OVA: OVA-sensitized mice treated by ASIT with OVA; ASIT with sOVA: OVA-sensitized mice treated by ASIT with sOVA; placebo: PBS-sensitized mice. (a) anti-OVA IgG1 response in mice with ASIT, (b) anti-OVA IgG2a response in mice with ASIT, (c) anti-OVA IgG response in mice with ASIT, (d) ratio of OVA-specific IgG1/IgG2a antibody in mice with ASIT, (*, p<0.05)</p

Histologic features of OVA- and sOVA-treated skin sites in BALB/c mice.

<p>(a) Mice sensitized with OVA and treated with PBS (“AD without ASIT”), (b) mice sensitized with OVA and treated with OVA (“ASIT with OVA”), (c) mice sensitized with OVA and treated with sOVA (“ASIT with sOVA”), and (d) mice sensitized with PBS (“placebo”). Skin sections were stained with H&E and examined at 100x. Scale bars 100 μm. There is marked hyperplasia of the epidermis, a dermal infiltrate (a-c). The cellular infiltrate consists of neutrophils, eosinophils, and lymphocytes. (e) Summary index of the main assessment criteria for histologic skin lesions.</p

Protocol of sensitization and ASIT with OVA or sOVA.

<p>Mice were sensitized with OVA (100 μg) or saline applied at 100 μl to a sterile patch. The patch was applied for 1-wk and then removed. ASIT was performed between the 1^st and 2^nd OVA applications by SC injection of increasing doses of OVA (a), sOVA (b), or PBS (as control). After ASIT, the mice had two 1-wk exposures to a patch separated by 2-wk intervals. The control group received PBS at the same time.</p