Oral administration of ovalbumin after sensitization attenuates symptoms in a mouse model of food allergic enteropathy

Oral immunotherapy (OIT) is a promising treatment of food allergy. To administer an appropriate oral dose of an allergenic component as OIT to individuals sensitized with a food allergen may prevent inducing food allergic inflammation in them. So we attempted to establish a mouse model to evaluate efficacy for oral administration of food allergen after sensitization. In BALB/c mice sensitized by injecting ovalbumin (OVA) with alum twice, OVA was administered before inducing inflammation by feeding the mice with egg white (EW) diet. Severe inflammatory responses, such as enteropathy, weight loss, IL-4 production, and increase of IgE antibody levels, were suppressed by administration with 4 mg of OVA 7 times before feeding EW diet. OVA administration alone induced a slight Th2 response, but no symptoms. The current study demonstrated that severe food allergic enteropathy could be prevented by pre-administration with appropriate dose of OVA to sensitized mice.     

The tick salivary protein, Salp15, inhibits the development of experimental asthma

Activation of Th2 CD4(+) T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 mug of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14-16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4(+) T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.     

The role of IL-13 in established allergic airway disease

The effectiveness of targeting IL-13 in models where airway hyperresponsiveness (AHR) and airway inflammation have already been established is not well-described. We investigated the effects of blocking IL-13 on the early and late phase airway responses and the development of AHR in previously sensitized and challenged mice. BALB/cByJ mice were sensitized (days 1 and 14) and challenged (days 28-30) with OVA. Six weeks later (day 72), previously sensitized/challenged mice were challenged with a single OVA aerosol and the early and late phase response and development of AHR were determined. Specific in vivo blockade of IL-13 was attained after i.p. injection of a soluble IL-13Ralpha2-IgG fusion protein (sIL-13Ralpha2Fc) on days 71-72 for the early and late responses and on days 71-73 for the development of AHR. sIL-13Ralpha2Fc administration inhibited the late, but not early, phase response and the OVA challenge-induced changes in lung resistance and dynamic compliance; as well, sIL-13Ralpha2Fc administration decreased bronchoalveolar lavage eosinophilia and mucus hypersecretion following the secondary challenge protocols. These results demonstrate that targeting IL-13 alone regulates airway responses when administrated to mice with established allergic airway disease. These data identify the importance of IL-13 in the development of allergen-induced altered airway responsiveness following airway challenge, even when administered before rechallenge of mice in which allergic disease had been previously established.     

Endotoxemia and hepatic injury in a rodent model of hindlimb unloading.

Hindlimb unloading (HU) is known to induce physiological alterations in various organ systems that mimic some responses observed after exposure to microgravity. In the present study, the effects of up to 4 wk of HU on the liver were assessed in male Wistar rats and two mouse strains: endotoxin-sensitive C57BL/6 mice and endotoxin-resistant C3H/HEJ mice. Plasma levels of endotoxin, a known stimulator of hepatic injury, were measured in portal and systemic blood samples. Endotoxin was elevated by approximately 50% in portal blood samples of mice and rats but was not detectable in systemic blood. This low-grade portal endotoxemia was associated with hepatic injury in rats and C57BL/6 mice as indicated by inflammation and elevated serum transaminase activities. Blood levels of the cytokine TNF-alpha were increased by approximately 50% in C57BL/6 mice; no significant elevation of this cytokine was detected in rats. Messenger RNA levels of the acute-phase proteins serum amyloid A, haptoglobin, and lipopolysaccharide binding protein were significantly enhanced after 3 wk of HU in endotoxin-sensitive rodents. In contrast, no histological changes or significant increases in serum enzyme activity were detected after HU in C3H/HEJ mice despite portal endotoxin levels of 222 +/- 83.4 pg/ml. At the 3-wk time point, expression of acute-phase proteins was not elevated in C3H/HEJ mice; however, expression after 4 wk of HU was similar to endotoxin-sensitive rodents. In conclusion, these findings indicate that HU induced mild portal endotoxemia, which contributed to the observed hepatic injury in endotoxin-sensitive rodents.     

IL-9 promotes but is not necessary for systemic anaphylaxis

Anaphylaxis represents an extreme form of allergic reaction, consisting of a sensitization phase during which allergen-specific IgE are produced and an acute effector phase triggered by allergen-induced degranulation of mast cells. We studied the role of IL-9, a Th2 cytokine implicated in asthma, in different models of murine anaphylaxis. Using a passive model of systemic anaphylaxis, in which anti-DNP IgE Abs were administered before challenge with DNP-BSA, we found that IL-9-transgenic mice or wild-type mice treated with IL-9 for 5 days were highly sensitive to fatal anaphylaxis. This effect was reproduced in both anaphylaxis-susceptible and -resistant backgrounds (FVB/N or [FVB/N x BALB/c] F(1) mice, respectively) and correlated with increased serum concentrations of mouse mast cell protease-1 level, a protein released upon mast cells degranulation. By contrast, IL-9 did not increase the susceptibility to passive cutaneous anaphylaxis. IL-9 expression also increased the susceptibility to fatal anaphylaxis when mice were sensitized by immunization against OVA before challenge with the same Ag. In this model, serum from sensitized, IL-9-transgenic mice was more potent in transferring susceptibility to OVA challenge into naive mice, indicating that IL-9 also promotes the sensitization stage. Finally, using IL-9R-deficient mice, we found that despite its anaphylaxis-promoting activity, IL-9 is dispensable for development of both passive and active anaphylaxis, at least in the C57BL/6 mouse background. Taken together, the data reported in this study indicate that IL-9 promotes systemic anaphylaxis reactions, acting at both the sensitization and effector stages, but is not absolutely required for this process.     

Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M(2) muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.     

P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma

Leukotriene E4 (LTE4) that plays a key role in airway inflammation is expressed on platelets and eosinophils. We investigated whether blocking of the P2Y12 receptor can suppress eosinophilic inflammation in a mouse model of asthma because platelets and eosinophils share this receptor to be activated. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. On each challenge day, clopidogrel, a P2Y12 antagonist was administered 30 min. before each challenge. Forty‐eight hours after the last OVA challenge, mice were assessed for airway hyperresponsiveness (AHR), cell composition and cytokine levels, including chemokine ligand 5 (CCL5), in bronchoalveolar lavage (BAL) fluid. EOL cells were treated with LTE4, with or without clopidogrel treatment, and intracellular and extracellular eosinophil cationic protein (ECP) expressions were measured to find the inhibiting function of P2Y12 antagonist on eosinophilic activation. The levels of P2Y12 expression were increased markedly in the lung homogenates of OVA‐sensitized and ‐challenged mice after platelet depletion. Administration of clopidogrel decreased AHR and the number of airway inflammatory cells, including eosinophils, in BAL fluid following OVA challenge. These results were associated with decreased levels of Th2 cytokines and CCL5. Histological examination showed that inflammatory cells as well as mucus‐containing goblet cells were reduced in clopidogrel‐administered mice compared to vehicle‐treated mice. Clopidogrel inhibited extracellular ECP secretion after LTE4 stimulation in EOL‐1 cells. Clopidogrel could prevent development of AHR and airway inflammation in a mouse model of asthma. P2Y12 can be a novel therapeutic target to the suppression of eosinophils in asthma.     

DA-9601, Artemisia asiatica herbal extract, ameliorates airway inflammation of allergic asthma in mice

We previously reported that DA-9601, ethanol herbal extract of Artemisia asiatica, inhibited histamine and leukotriene releases in guinea pig lung mast cells activated with specific antigen/antibody reaction. This study aimed to evaluate the inhibitory effect of DA-9601 on the OVA-induced airway inflammation in allergic asthma mouse model. BALB/c mice were sensitized and challenged with OVA. DA-9601 was administered orally 1 h before every local OVA-challenge. OVA-specific serum IgE was measured by ELISA, recruitment of inflammatory cells in BAL fluids and lung tissues by Diff-Quik and H&E staining, respectively, the expressions of CD40, CD40L and VCAM-1 by immunohistochemistry, goblet cell hyperplasia by PAS staining, activities of MMPs by gelatin zymography, expressions of mRNA and proteins of cytokines by RT-PCR and ELISA, activities of MAP kinases by western blot, and activity of NF-KappaB by EMSA. DA-9601 reduced IgE level, recruitment of inflammatory cells into the BAL fluid and lung tissues, expressions of CD40, CD40L and VCAM-1 molecules, goblet cell hyperplasia, MMPs activity, expressions of mRNA and productions of various cytokines, activities of MAP kinases and NK-KappaB increased from OVA-challenged mice. These data suggest that DA-9601 may be developed as a clinical therapeutic agent in allergic diseases due to suppressing the airway allergic inflammation via regulation of various cellular molecules expressed by MAP kinases/NF-KappaB pathway.     

Identification of suberization-associated anionic peroxidase as a possible allergenic protein from tomato

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.     

Early phase bronchoconstriction in the mouse requires allergen-specific IgG

Allergen provocation of allergic asthma patients is often characterized by an initial period of bronchoconstriction, or early phase reaction (EPR), that leads to maximal airway narrowing within 15-30 min, followed by a recovery period returning airway function to baseline within 1-2 h. In this study, we used a defined OVA provocation model and mice deficient for specific leukocyte populations to investigate the cellular/molecular origins of the EPR. OVA-sensitized/challenged wild-type (C57BL/6J) mice displayed an EPR following OVA provocation. However, this response was absent in gene knockout animals deficient of either B or T cells. Moreover, transfer of OVA-specific IgG, but not IgE, before the OVA provocation, was capable of inducing the EPR in both strains of lymphocyte-deficient mice. Interestingly, an EPR was also observed in sensitized/challenged mast cell-deficient mice following an OVA provocation. These data show that the EPR in the mouse is an immunologically based pathophysiological response that requires allergen-specific IgG but occurs independent of mast cell activities. Thus, in the mouse the initial period of bronchoconstriction following allergen exposure may involve neither mast cells nor IgE-mediated events.     

耐受性树突状细胞预防和治疗卵清蛋白过敏小鼠的的研究

目的 比较腺病毒重组CTLA4Ig/α4β7诱导的耐受性树突状细胞对卵清蛋白(OVA)致敏小鼠的预防和治疗效果.方法 BALA/c小鼠32只,分为4组.基础致敏24h后回输耐受性树突状细胞为预防组;肠道激发4h后回输耐受性树突状细胞为治疗组;回输生理盐水为阴性对照组和OVA过敏小鼠为阳性对照组.观察各组小鼠过敏症状缓解情况;HE染色观察空肠形态;甲苯胺兰染色观察小肠固有层肥大细胞聚集及脱颗粒现象;ELISA测定各组血清中OVA特异性IgE水平.结果 与阳性对照组相比,治疗组小鼠OVA特异性IgE水平显著降低(0.25±0.05 vs 0.17±0.03) (P ＜0.05);体重增长明显(3.16 g±0.75 g vs 5.04 g±0.49 g)(P ＜0.05);腹泻症状减轻;HE染色可见治疗组小肠绒毛中上皮细胞局灶性坏死、脱落,固有层炎症细胞浸润现象明显改善;肥大细胞聚集和脱颗粒现象改善.而预防组较阳性对照组过敏症状、小肠形态学及OVA特异性IgE水平差异均无统计学意义.结论Ad CTLA4Ig/Adα4β7修饰的致耐受性树突状细胞对卵清蛋白过敏小鼠具有治疗作用而无预防作用.     

10株乳酸菌黏附小鼠派伊尔结及免疫调节作用研究

目的探索乳酸菌黏附派伊尔结效率与其免疫调节作用的关系。方法利用荧光定量分析法测定10株乳酸菌黏附派伊尔结的能力,利用乳酸菌与派伊尔结组织块共培养测定乳酸菌调节派伊尔结细胞因子释放活性。选择诱导细胞因子谱相似、黏附派伊尔结性质不同的菌株进行动物实验,测定其对卵清蛋白（OVA）致敏小鼠粪便中分泌型免疫球蛋白A（sIgA）以及腹腔巨噬细胞吞噬活性的调节作用。结果10株乳酸菌黏附派伊尔结的性质具有菌株依赖性,其中L.rhamnosusGG、L.acidophilusFn037、L.plantarumFn008和L.caseiFn012黏附性依次降低,但诱导派伊尔结TNF-α、IL-10和IL-12释放的性质相同。这4株菌增强OVA致敏小鼠腹腔巨噬细胞吞噬活性和升高粪便sIgA的能力与其黏附性相关。结论乳酸菌黏附派伊尔结是决定其免疫调节活性的主要因素之一。     

In situ amplification of 5-lipoxygenase and 5-lipoxygenase-activating protein in allergic airway inflammation and inhibition by leukotriene blockade

Leukotrienes are important mediators of the eosinophilic influx and mucus hypersecretion in the lungs in a murine model of asthma. We used in situ PCR in this model of human asthma to detect lung mRNA for 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP), key proteins necessary for leukotriene synthesis. Lung tissue was obtained on day 28 from mice treated with i.p. (days 0 and 14) and intranasal (days 14, 25, 26, and 27) OVA or saline. After fixation, the tissue sections underwent protease- and RNase-free DNase digestion, before in situ RT-PCR using target-specific cDNA amplification. 5-LO and FLAP-specific mRNA was visualized by a digoxigenin detection system, and positive cells were analyzed by morphometry. 5-LO and FLAP-specific mRNA and protein were associated primarily with eosinophils and alveolar macrophages in the airways and pulmonary blood vessels in OVA-sensitized/challenged mice. 5-LO and FLAP protein expression increased on a per-cell basis in alveolar macrophages of OVA-treated mice compared with saline controls. Pulmonary blood vessel endothelial cells were also positive for 5-LO, FLAP mRNA, and protein. 5-LO inhibition significantly decreased 5-LO and FLAP-specific mRNA and protein expression in the lung inflammatory cells and endothelial cells. These studies demonstrate a marked increase in key 5-LO pathway proteins in the allergic lung inflammatory response and an important immunomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.     

Nitration of the egg-allergen ovalbumin enhances protein allergenicity but reduces the risk for oral sensitization in a murine model of food allergy

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.     

Absorption of a protein gavage in Zucker lean rats. Influence of protein content in the diet.

The rate of protein absorption was measured in Zucker lean rats. Rats were fed with a bolus that contained ca. 300 mg of 14C-labelled protein at the beginning of the light cycle. Blood was extracted from the portal vein at intervals up to 9 hours after gavage. Label incorporation into tissue protein was monitored. The digestion and absorption of protein was slow, and 9 hours after the gavage, 20% of the bolus remained in the stomach. Forty percent of the protein was absorbed in the first hour. This was followed first by a linear absorption process, then by the amino acid incorporation into tissue proteins. The appearance of label in the portal vein increased progressively for up to four hours, shifting to a progressive decrease that coincides with the maintenance of this label in the tissues. The skin, the striated muscle and the liver showed the highest amounts of labelled proteins. The application of this model to animals fed low-(LP) or high-protein (HP) content diets showed that the HP group digested the protein faster than the LP group, and that catabolism of the amino acids was higher in the HP group. The LP group digested protein much more slowly than the RD (control) group, but protein accretion was more efficient.     

Identification of alternative pathway serum complement activity in the blood of the American alligator (Alligator mississippiensis)

Incubation of different dilutions of alligator serum with sheep red blood cells (SRBCs) that had not been sensitized with antibodies resulted in concentration-dependent hemolytic activity. This hemolytic activity was not affected by the presence of ammonium hydroxide and methylamine, known inactivators of the classical complement cascade. However, the hemolytic activities were inhibited by EDTA and salicylaldoxime, indicating that the alternate pathway is primarily responsible for these activities. Immunofixation of electrophoretically-resolved alligator serum proteins with antihuman C3 polyclonal antibodies resulted in detection of a protein antigenically similar to human C3 in alligator serum. SDS-PAGE, followed by Western blot analysis, revealed the presence of two alligator serum proteins with nearly identical molecular weights as human C3alpha and C3beta. SRBC hemolysis and antibacterial activity by alligator serum was significantly reduced in the presence of antihuman C3 antibodies. The hemolytic effect of alligator serum was shown to occur rapidly, with significant activity within 5 min and maximal activity occurring at 15 min. SRBC hemolysis was also temperature-dependent, with reduced activity below 15 degrees C and above 30 degrees C. These data suggest that the antibiotic properties of alligator serum are partially due to the presence of a complement-facilitated humoral immune response analogous to that described in mammalian systems.     

Impact of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis in mice

The effects of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis (WDEIA) were investigated by using a mouse model. The gliadin fraction was prepared as a 70% ethanol-soluble solution, and ω-5 gliadin was purified by chromatography. Purified ω-5 gliadin was run on SDS-PAGE gel to reveal three bands with a molecular mass of 53-60 kDa and had the characteristic N-terminal sequence of ω-5 gliadin. The mice were sensitized to the gliadin fraction, and the anaphylactic response was assessed by measuring the body temperature and voluntary physical activity. An oral administration of ω-5 gliadin evoked a significant drop in both the body temperature and voluntary physical activity, similar to the effects of the whole gliadin fraction. ELISA and immunoblotting analyses revealed that the IgE expression from sensitized mice reacted most strongly to ω-5 gliadin. Taken together, these results indicate ω-5 gliadin to be a major allergen responsible for stimulating WDEIA in mice, with the characteristic potential for stimulating IgE production.     

Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse model of asthma

In vitro and in vivo studies, in both animal models and human asthmatics, have implicated IL-4 as an important inflammatory mediator in asthma. In a murine asthma model, we examined the anti-inflammatory activities of soluble IL-4R (sIL-4R). In this model, mice sensitized to OVA by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The OVA challenge elicits an eosinophil infiltration into the lungs, with widespread mucus occlusion of the airways, and results in bronchial hyperreactivity. sIL-4R (0.1-100 microgram) was administered by either i.n. or i.p. routes before OVA challenge in OVA-sensitized mice. Both blood and bronchoalveolar lavage fluid levels of sIL-4R were significantly elevated compared with controls by i.n. delivery of 100 microgram sIL-4R; i.p. delivery of 100 microgram sIL-4R only raised blood levels of sIL-4R. The i.n. administration of 100 microgram sIL-4R before allergen challenge significantly reduced late phase pulmonary inflammation, blocking airway eosinophil infiltration, VCAM-1 expression, and mucus hypersecretion. In contrast, i.p. delivery of 100 microgram sIL-4R inhibited only the influx of eosinophils into the lungs, but not airway mucus release. Furthermore, sIL-4R treatment by either i.n. or i.p. routes did not reduce airway hyperreactivity in response to methacholine challenge. Thus, elevating airway levels of sIL-4R through the administration of exogenous sIL-4R is effective in blocking the late phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that sIL-4R may have beneficial anti-inflammatory effects in asthmatic patients.     

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.