BACKGROUND:We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins.OBJECTIVE:The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.DESIGN: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.RESULTS: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.CONCLUSION: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.

Background Impairment of gastric digestion due to pH elevation increases the risk for food allergy induction. As patients after Roux-en-Y gastric bypass (RYGB) surgery have lower gastric acidity and less gastric gland secretion, we aimed to analyse in a prospective study the effect of limiting gastric digestion capacity by surgical intervention on the immune response towards allergens. Methods Nine patients undergoing RYGB surgery for morbid obesity and one control patient having undergone surgery for treatment of an incisional hernia were enrolled in the study. Before and 1, 3, 6, 9 and 12 months after surgery, blood was collected for analysis of specific IgE antibodies, and patients were subjected to skin prick testing with 16 food and 18 aeroallergens. Results Skin prick test results revealed an increase of positive reactions indicating sensitisations towards the tested food and aeroallergens in 77.8 and 88.9 % of the patients, respectively, after surgical elimination of gastric digestion. These results were in line with elevated titers of food- and aeroallergen-specific IgE antibodies in 7 out of 9 (7/9) and 5/9 patients, respectively, after RYGB surgery. Serum cytokine levels revealed a mixed response for IFN-γ and were mostly beneath detection limit for IL-4. Conclusion A change of IgE reactivity pattern occurred after impairment of gastric digestion due to surgical elimination underlining the important gastric gatekeeping function during oral sensitisation. Even though this study indicates an increased allergy risk for gastric bypass patients, further studies are needed to investigate in-depth the immunological changes associated with RYGB surgery.

Methods BALB/c mice were nasally sensitized 5 times in biweekly intervals with KLH alone or with KLH in conjunction with acrolein. Airway hyperreactivity was was measured according to change of enhanced pause and KLH-specific anaphylactic reaction was monitored in vivo Levels of specific antibodies as well as cytokine profile of KLH-stimulated splenocytes were analyzed by ELISA. Further, mouse D2F2-tumor cells were grafted to the flanks and tumor growth monitored in mice previously exposed to acrolein or buffer.

Background Recently, we demonstrated in an experimental mouse study that mucosal M-cell targeting with Aleuria aurantia lectin (AAL) coated Poly(D,L-lactide-co-glycolide) (PLGA) microspheres represents a promising oral treatment approach in IgE mediated allergy. Due to its structural similarities with AAL we aimed to assess Neuraminidase (NA) from Vibrio cholerae as a novel M-cell specific targeters and compared its properties to AAL and wheat germ agglutinin (WGA) representing 2 plant lectins, which target either M-cells or epithelial cells, respectively. Methods The resistance against gastric digestion of NA, AAL and WGA was analyzed in simulated gastric fluid (SGF) experiments. Intestinal epithelial binding was determined using the colon carcinoma cell line Caco2, which represents a well established model for the human intestinal epithelium. Binding specificity was evaluated by inhibition experiments by incubating Caco2 cells with Biotin-labeled NA, AAL or WGA, after preincubation with a-L fucose, monoganglioside (GM1) or N,N′,N″-triacetyl-chitotriose (TCT). The stimulatory effects of the targeting substances on the intestinal microenvironment were investigated by cytokine read-out experiments in real-time PCR. Further, the transeptihelial uptake of NA-, AAL- or WGA-functionalized fluospheres was evaluated in a human M-cell co-culture model. Results All 3 targeters were stable up to 180 minutes in SGF, indicating their suitability for oral application. The binding partners were a-L fucose for AAL and TCT for WGA, whereas NA interacts with intestinal epithelial cells via a-L fucose and additionally GM1. NA skewed the cytokine production by inducing a 2-fold increase of the Th1 cytokine IFNg after 60 minutes, whereas AAL decreased the overall cytokine expression. In a human M-cell co-culture model, a higher transepithelial transport rate of fluospheres coated with NA and AAL was observed as compared to WGA and plain particles. Conclusions NA specifically targets M-cells via a-L fucose and additionally GM1 and, thus, increases the transepithelial transport of NA coated particles. Due to the immunomodulatory capacity on intestinal epithelial cells, NA functionalized microspheres may represent a promising M-cell specific targeting approach for oral immunotherapy.

Background: Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy.Objective: We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy.Methods and results: Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1(-/-) and SphK2(-/-) mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4(+) effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals.Conclusion: Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy. (C) 2011 Elsevier B.V. All rights reserved.

Methods and results BALB/c mice were fed untreated (OVA), sham-nitrated (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze systemic effects, mice were injected the allergens intraperitoneally (i.p.). Animals being fed OVA or snOVA with antiacids developed elevated IgE, IgG1 and IgG2a titers. Oral immunizations of nOVA under acid-suppression did not result in IgG and IgE formation. However, all i.p. immunized mice revealed high levels of IgE, which were significantly increased in the group being injected nOVA. In RBL-assays, all groups with OVA-specific IgE showed a significant increased mediator release with nOVA as trigger compared to OVA. To analyze the immune response in the involved organ, gastric tissues were screened for cytokine expression by real-time-PCR. Only the acid-suppressed groups being fed OVA or snOVA revealed a higher expression of Th2 and inflammatory markers. In gastric digestion experiments, nOVA was degraded within minutes, whereas OVA and snOVA remained stable up to 120min. Additionally, HPLCchip-MS/MS analysis revealed the most efficiently nitrated tyrosine residue within an ovalbumin epitope recognized exclusively after oral immunization.

Background Nitration of proteins on tyrosine residues, which can occur due to polluted air under "summer smog" conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. Methodology/Principal Findings BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y107) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. Conclusions/Significance These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.