Nitrated BLG enhances the anaphylactic response in BLG immunized animals.

<p>After i.v. allergen challenge all BLG immunized groups showed significantly elevated levels of the anaphylaxis marker mMCP-1 compared to naïve control groups (A). Allergic mice injected with nitrated BLG (BLGn) revealed a significantly higher mMCP-1 release compared to mice only receiving untreated (BLG) or sham-nitrated (BLGs) BLG. Systemic challenge of allergic mice with different preparations of OVM did not result in differences of mMCP-1 between OVM, OVMs and OVMn (B). Mouse MCP-1 concentrations were compared with One-way ANOVA. Body temperature of all groups was assessed before and 15 and 30 min after i.v. challenge (C, D). A significant drop of body temperature was observed in all allergic mice. Animals challenged with BLGn showed a significant decline of body temperature compared to BLG and BLGs, while in OVM allergic mice decrease was most prominent in OVMs. Mean temperatures of all groups at 30 min were compared using One-way ANOVA prior to Tukey multiple comparison test (*P>0.05, **P<0.01, ***P<0.001). BLG, beta-lactoglobulin; BLGn, nitrated BLG; BLGs, sham-nitrated BLG; i.v., intravenous; mMCP-1, mouse mast cell protease-1; OVM, ovomucoid; OVMn, nitrated OVM; OVMs, sham-nitrated OVM</p

Increased dimerization of nitrated BLG.

<p>(A) SDS-PAGE analysis revealed an enhanced dimerization of nitrated BLG compared to untreated and sham-nitrated proteins. (B) OVM formed dimers independent of the nitration status. BLG, beta-lactoglobulin; M, marker lane; OVM, ovomucoid</p

Induction of allergen specific IgE, IgA, IgG1 and IgG2a in immunized mice.

<p>Immunization elicited equally elevated specific IgE titers in mice being injected with the same allergen (A, BLG; B, OVM) whereas naïve control groups did not show any allergen specific IgE antibody production. BLG (C, E, G) and OVM (D, F, H) allergic mice showed significantly higher levels of allergen specific IgG1, IgG2a and IgA compared with naïve mice. Significantly lower levels of specific IgG2a were observed in the group i.v. challenged with nitrated BLG (BLGn). Significantly lower levels of specific IgG1 were found in the group i.v. challenged with nitrated OVM (OVMn). Independent of the isotype, higher antibody levels were found in mice sensitized with BLG compared to OVM sensitized animals. Data obtained for IgG2a of OVM allergic mice were transformed by applying the logarithm to base 10. Antibody levels of groups were compared at the time-point of sacrifice with One-way ANOVA followed by Tukey post test. (**P<0.01, ***P<0.001) BLG, beta-lactoglobulin; BLGn, nitrated BLG; i.v., intravenous; OVM, ovomucoid; OVMn, nitrated OVM</p

Nitrated BLG reveals an altered secondary structure.

<p>Samples originally dissolved in 10 mM Na₂HPO₄ buffer were diluted to 0.1 mg/ml in ultrapure distilled water according to the following ratios for: BLGs (1/21.55), BLGn (1/14.98), OVMs (1/16.20), OVMn (1/17.84). For nitrated BLG (A) an alteration of the secondary structure was observed as indicated by changed spectra, which was not observed for OVM (B). Results are expressed as the mean residue ellipticity Θ (y-axis) and plotted against the respective wavelength (x-axis). BLG, beta-lactoglobulin; OVM, ovomucoid</p

Nitration of β-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice

<div><p>Background</p><p>We revealed in previous studies that nitration of food proteins reduces the risk of <i>de novo</i> sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, <i>in vitro</i> experiments suggested an increased capacity of effector cell activation by nitrated food proteins.</p><p>Objective</p><p>The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.</p><p>Design</p><p>BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood.</p><p>Results</p><p>A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation.</p><p>Conclusion</p><p>Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.</p></div

Nutrients / Immune Effects of the Nitrated Food Allergen Beta-Lactoglobulin in an Experimental Food Allergy Model

Food proteins may get nitrated by various exogenous or endogenous mechanisms. As individuals might get recurrently exposed to nitrated proteins via daily diet, we aimed to investigate the effect of repeatedly ingested nitrated food proteins on the subsequent immune response in non-allergic and allergic mice using the milk allergen beta-lactoglobulin (BLG) as model food protein in a mouse model. Evaluating the presence of nitrated proteins in food, we could detect 3-nitrotyrosine (3-NT) in extracts of different foods and in stomach content extracts of non-allergic mice under physiological conditions. Chemically nitrated BLG (BLGn) exhibited enhanced susceptibility to degradation in simulated gastric fluid experiments compared to untreated BLG (BLGu). Gavage of BLGn to non-allergic animals increased interferon- and interleukin-10 release of stimulated spleen cells and led to the formation of BLG-specific serum IgA. Allergic mice receiving three oral gavages of BLGn had higher levels of mouse mast cell protease-1 (mMCP-1) compared to allergic mice receiving BLGu. Regardless of the preceding immune status, non-allergic or allergic, repeatedly ingested nitrated food proteins seem to considerably influence the subsequent immune response.(VLID)458847