Kinetic and equilibrium constants for complexes formed by α-casein, α subunit of β-conglycinin (α), α-T and PA with the 1D5 mAb, specific for α-casein.

<p><i>k_ass</i>: Association constant, <i>k_diss</i>: Dissociation constant, K_A: Affinity constant, K_D: Equilibrium Dissociation constant.</p

Inflammatory response induced after nasal delivery of U-Omp16.

<p>C57BL/6 mice (<i>n</i>=5/group) were intranasally administered with U-Omp16 or PBS as control and the number of total cells (<b>A</b>) and differential counts of macrophages (<b>B</b>), neutrophils (<b>C</b>) and lymphocytes (<b>D</b>) were determined in BAL at different time points. (<b>E</b>) Animals were intranasally administered with OVA_(AF647) alone or OVA_(AF647) plus U-Omp16. Lungs were obtained at different time points after delivery and the emission of fluorescence was evaluated in cell suspensions from each lung (1 x10⁶ cells). (<b>F</b>) C57BL/6 animals were administered through the nasal route with U-Omp16 or PBS as control and at different times post administration the level of TNF-α, IL-10 in lung tissues, and (<b>G</b>) TNF-α, IL-10, CCL2, CCL3 and CCL5 in BAL were determined by ELISA. Samples were assayed in duplicated and data represent the mean ±SEM from each group of five mice, (**<i>P</i><0.001, *<i>P</i><0.05 <i>vs</i> PBS group). These results are representative of 3 independent experiments with similar results.</p

U-Omp16 administrated <i>in</i><i>vivo</i> modulates the allergic reaction in a mouse model of food allergy.

<p>(<b>A</b>) Outline of the experimental design for the mouse model of food allergy in BALB/c mice (<i>n</i>=10/group). (<b>B</b>) Hypersensitivity scores of sensitized and sensitized/treated mice 30 minutes following the i.g. challenge with CMP (<i>n/group=10</i>). (<b>C</b>) Delayed-type hypersensitivity (DTH) response to CMP was assayed 3 weeks after the last boost to evaluate the cellular immune response <i>in vivo</i>. Twenty µg of CMP were injected into one footpad, and saline was injected into the contra lateral footpad, as a negative control. The thickness of both footpads was measured 48 h later. (<b>D</b>) Cutaneous test in sensitized and treated mice to evaluate the induction of immediate inflammation. (<b>E</b>) Determination of CMP-specific serum IgG1, IgG2a and IgE after oral CMP sensitization and intranasal treatment. (<b>H</b>) Treatment with U-Omp16 stimulates the induction of CD4⁺ CMP-specific T cells that produce IFN-γ. Spleen cells from CMP sensitized and treated mice were stained with specific anti-CD4 (PE) monoclonal Ab. After permeabilization cells were stained with anti-IFN-γ (FITC) or isotype control (FITC) monoclonal Abs for intracellular flow cytometry analysis. (<b>G</b>) Splenocytes were collected 24 h after the oral challenge and stimulated <i>in vitro</i> with CMP (350 µg/ml) or casein (200 µg/ml) for 72 h. Levels of IL-5 in culture supernatants of spleen cells from sensitized and treated mice were determined by ELISA. (<b>I</b>) mRNA expression for cytokines (IL-5 and IFN-γ) and transcription factors (t-bet and gata-3) was quantified 24 h after oral challenge in jejunum segments. Data are expressed as mean values ±SEM (<i>***P</i><0.001<i>, **P</i><0.01, <i>*P</i><0.05 <i>vs</i> CMP treated group). These results are representative of two independent experiments with similar results.</p

Schematic localization of the identified spots along the α-T sequence and sequence alignment analysis.

<p><b>A</b>) Analysis of the sequence alignment of α-T with IgE epitopes in bovine caseins (IEDB) shown as hit distribution of amino acid similitude per position along the sequence of α-T. Positions of α-T with high hit values (black columns) correlate with positive spots of the overlapping assay (spots 9, 10 and12); <b>B</b>) The sequence of α-T (1–234 aa) is depicted along with overlapped peptides, PA and PN (305–539 aa correspond to α). <i>In silico</i> predicted alpha-helix (white ovals) and beta-sheet (black arrows) zones are indicated. PA contains the amino acid positions with the highest hits and the α-helix and β strand secondary structures.</p

<i>In vivo</i> cross-reactivity assessed in a food allergy mouse model.

<p><b>A</b>) Schematic representation of the experimental protocol: BALB/c mice were subjected to weekly intragastric sensitization with cholera toxin and CMP from day 0 through day 35. Challenge was performed at day 45 by ig administration of proteins (CMP, SP or OVA). Control mice only received CMP and then they were challenged with the different antigens. Symptoms were observed 30 min following the oral challenge and scored according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082341#pone-0082341-t002" target="_blank">Table 2</a>. <b>B</b>) Clinical scores assigned to symptoms observed 30 min following the sublingual challenge with 10 µg of β-lg, α, α-T, PA or OVA. <b>C</b>) Specific IgE and IgG1 in serum of milk-sensitized and control mice were assessed by EAST or indirect ELISA at day 45, respectively (mean values ± SEM). <b>D</b>) Cutaneous test performed in sensitized mice subcutaneously injected with 10 µg of antigens (α, α-T or PA) in the right flank and with saline in the left flank. The presence of blue color in the skin within minutes was considered a positive cutaneous test. <b>E</b>) Levels of IL-5 and IFN-γ assayed on stimulated splenocyte supernatants by ELISA (mean values ± SEM). Results correspond to a single experiment with five mice per condition, representative of three separate experiments that gave similar results. Statistically significant differences are denoted as starred values (*) ***p<0.005, **p<0.01, *p<0.05.</p

U-Omp16 induces a T helper 1 immune response when administered as nasal adjuvant.

<p>C57BL/6 mice were immunized by the nasal route with: OVA plus (i) PBS or ii) U-Omp16 previously digested with proteinase K (U-Omp16PK) or plus (iii) U-Omp16. Three weeks after last immunization animals were sacrificed and spleen cells were stimulated <i>in vitro</i> with OVA 500 µg/ml or complete medium (RPMI). Culture supernatants were harvested 5 days after stimulation and cytokine concentration of (<b>A</b>) IFN-γ, (<b>B</b>) IL-17, (<b>C</b>) IL-4 and (<b>D</b>) IL-10 (pg/ml) were determined by ELISA. U-Omp16 when administered as nasal adjuvant stimulates the induction of CD4⁺ and CD8⁺ OVA-specific T cells that produce IFN-γ. Percentages are represented for spleen (<b>E</b>) CD8⁺ or CD4⁺ T lymphocytes, and lung CD8⁺ T cells (<b>F</b>) expressing IFN-γ. (<b>G</b>) Anti-OVA IgG was determined in sera from immunized animals on days 0 (pre-immune) and 30 (post-immune) by indirect ELISA. Data represent the mean ±SEM from each group of five mice; (***<i>P</i><0.001, <i>**P</i><0.01, <i>*P</i><0.05 <i>vs</i> OVA group). These results are representative of 3 independent experiments with similar results.</p

Sequential competitive ELISA with the CMP-specific rabbit polyclonal antiserum and 1D5 mAb.

<p>Microtiter plates were coated with CMP (1 µg/well) (<b>A</b> and <b>C</b>), α (0.1 µg/well) or α-T (0.1 µg/well) (<b>B</b>). CMP-specific polyclonal antiserum (<b>A</b>: 1∶200,000; <b>B</b>: 1∶6,000) or α-casein-specific mAb (<b>C</b>: 1∶300,000) were pre-incubated with different inhibitors at several concentrations and then added to Ag-coated wells. Then the appropriate conjugated secondary antibodies were added and the color was developed.</p

<i>In silico</i> and <i>in vitro</i> analysis of α, α-T and PA.

<p><b>A</b>) Homology model obtained using the Modeller and visualized by PyMOL of α-subunit of β-conglycinin, α-T and PA, based on NCBI Conserved Domain Search results with α′ subunit as template (α-helices are shown in blue and β-sheets as dark grey arrows on the structure). <b>B</b>) Surface representation of positive overlapped peptides marked in different colors according to the intensity of the reaction (from pale yellow to red); I and II correspond to opposite sides of the α molecule. Positive peptides localized on the surface of the molecule seem to converge in three regions or putative epitopes. The remaining part of the molecule is represented in transparent spheres showing the secondary structure. <b>C</b>) SDS-PAGE of recombinant PA and PN and immunoblotting developed with 1D5 α-casein-specific mAb and a representative serum of CMA patients. Alpha-s1 casein was used as positive control (Molecular masses are shown on the left as kDa). <b>D</b>) Dot-blot of recombinant treated and untreated PA revealed with a pool of CMA patient sera, α-casein-specific mAb and CMP-specific polyclonal rabbit antiserum. Denaturing conditions consisted on: 0.1M DTT solution, 6M urea or 100°C for 10 minutes. <i>Cas: Alpha-s1 casein</i>.</p

Clinical scores assigned to symptoms triggered following the oral or sublingual challenge.

<p>Clinical scores assigned to symptoms triggered following the oral or sublingual challenge.</p

Immunochemical cross-reactivity.

<p><b>A) Detection of specific human serum IgE antibodies by indirect ELISA.</b> Microtiter plates were coated with CMP, SP, recombinant α-subunit of β-conglycinin “α” or α-T, and sequentially incubated with sera of CMA patients and anti-human IgE specific conjugate. 15/15 sera contained IgE antibodies specific for CMP, 13/15 sera contained IgE antibodies specific for α and 14/15 sera contained IgE antibodies for α-T. <b>B) SDS-PAGE and immunoblotting of CMP and SP.</b> SDS-PAGE was performed under non reducing conditions for CMP (<b>upper panel</b>), SP (<b>middle panel</b>), and purified recombinant α and the α-T fraction (<b>lower panel</b>). Immunoblottings were developed with patient sera containing milk-specific IgE antibodies or control sera (from non-allergic patients) (1∶4), CMP-specific rabbit polyclonal antiserum (1∶1,000), and α-casein-specific monoclonal antibody (1D5 mAb 1∶1,500). <i>CMP: Cow's milk protein, cas: caseins, SP: soybean protein, G: 11S glycinin subunits; β-conglycinin subunits are indicated as α, α′ and β. M: Molecular masses are given on the left in kilo Daltons (kDa).</i></p