Food-allergic Subjects Research Articles

Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children

Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges). We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker. Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in invitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays. We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA exvivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3+ cells over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3+ cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3+ cells were also more clonally expanded than the FOXP3- population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3+ cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127-CD25+ cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for TH2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of TH2 cytokines and pathogenic TH2/T follicular helper markers. Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3+ cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.

Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

ASSOCIATION OF STAT6 GENE SINGLE NUCLEOTIDE POLYMORPHISM WITH FOOD ALLERGY

Abstract Background Food allergy (FA) may be IgE-mediated, non-IgE-mediated or mixed. Genetic predispositions result in dysregulation of the immune system and lead to FA when exposed to environmental triggers. The transcription factor STAT6 is activated by IL-4 and IL-13 and activates cytokine signaling in immune and target tissue cells; contributing in the pathogenesis of allergic disorders including FA. The rs324015 single nucleotide polymorphism (SNP) in the STAT6 gene was previously associated with nut and cow’s milk allergy. Objective to investigate the association of the rs324015 STAT6 gene SNP with food allergy. Patients and Methods After ethical committee approval and informed consent from the patients, this retrospective case control study was conducted on 100 Egyptian children divided into two groups. Group I consisted of 50 children with positive history of FA supported by positive skin prick test +/- oral food challenge and group II consisted of 50 healthy age-matched controls. Blood samples were collected and genotyped using Taqman® Realtime PCR assays. Results We found that the homozygous G genotype and G allele of rs324015 were significantly higher among food allergic subjects than controls. We report no effect of any specific genotype on the phenotype of allergic disease or reacting to a specific food more than others. Genotypes were also not related to disease severity, eosinophil counts, total IgE levels or a family history of atopic disease. Conclusion rs324015 STAT6 gene SNP may be involved in the pathophysiology of food allergy.

New Insights on the Relation Between Human Microbiota and Food Allergy

Food allergy onset occurs in early life, in fact an Australian non-selected 1-year-old children cohort showed a prevalence of sesame, peanut and egg allergy of 8.9, 3.0 and 0.8 % respectively. Other experiments showed that clinical reactivity to such foods arises around 4 months of age. Several researchers reported dysbiosis in food allergic subjects. Some studies about human microbiome genetics evidenced altered gut microbiota in individuals suffering from both cow’s milk and egg allergies. It is generally accepted that dysbiosis arises prodromal to the food allergy onset. In fact, after analyzing gut microbiota in children, belonging to a Canadian cohort, within 12 months of age, it was found proof of food sensitization after 12 months of age. The aim of this opinion paper is to enforce existing evidences and, above all, to introduce the concept that, in many cases, human features and perceptions are mostly shaped by microbiota, as outlined in my previous papers.

Fecal microbiome signatures are different in food-allergic children compared to siblings and healthy children.

Intestinal microbes have been shown to influence predisposition to atopic disease, including food allergy. The intestinal microbiome of food-allergic children may differ in significant ways from genetically similar non-allergic children and age-matched controls. The aim was to characterize fecal microbiomes to identify taxa that may influence the expression of food allergy. Stool samples were collected from children with IgE-mediated food allergies, siblings without food allergy, and non-allergic controls. Stool microbiome characterization was performed via next-generation sequencing (Illumina) of the V1V3 and V4 variable regions of the 16S rRNA gene. Bacterial diversity, evenness, richness, and relative abundance of the operational taxonomic units (OTUs) were evaluated using QIIME. ANOVA and Welch's t test were utilized to compare groups. Sixty-eight children were included: food-allergic (n=22), non-food-allergic siblings (n=25), and controls (n=21). When comparing fecal microbial communities across groups, differences were noted in Rikenellaceae (P=.035), Actinomycetaceae (P=.043), and Pasteurellaceae (P=.018), and nine other distinct OTUs. Food-allergic subjects had enrichment for specific microbes within the Clostridia class and Firmicutes phylum (Oscillobacter valericigenes, Lachnoclostridium bolteae, Faecalibacterium sp.) compared to siblings and controls. Identification of Clostridium sp. OTUs revealed differences in specific Clostridia drive the separation of the allergic from the siblings and controls. Alistipes sp. were enriched in non-allergic siblings. Comparisons in the fecal microbiome of food-allergic children, siblings, and healthy children point to key differences in microbiome signatures, suggesting the role of both genetic and environmental contributors in the manifestation of food-allergic disease.

A procedure for grouping food consumption data for use in food allergen risk assessment

Food allergic subjects need to avoid the allergenic food that triggers their allergy. However, foods can also contain unintended allergens. Food manufacturers or authorities need to perform a risk assessment to be able to decide if unintended allergen presence constitutes a risk to food allergic consumers. One of the input parameters in risk assessment is the amount of a given food consumed in a meal. There has been little emphasis on how food consumption data can be used in food allergen risk assessment. The aim of the study was to organize the complex datasets from National Food Consumption Surveys from different countries (France, Netherlands and Denmark) to be manageable in food allergen risk assessment. To do this, a two-step method was developed. First, based on initial groups of similar food items, the homogeneity of consumption was evaluated using a customized clustering method. Then, the risk was calculated for each initial food group and its subgroups to verify if it also represents a relevant difference in risk. Forty-eight food groups were designated in Denmark (53 in the Netherlands, 54 in France). Finally, summary statistics and names for each food group for the Danish data illustrate the results when applying the procedure.

Identification and characterization of DC-SIGN-binding glycoproteins in allergenic foods.

DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) is a C-type lectin receptor expressed on macrophages and dendritic cells. DC-SIGN has high affinity for fucosylated glycans in several plant glycoproteins and pathogens. DC-SIGN is thought to be crucial for the development of allergic sensitization. However, the precise role of DC-SIGN in food allergy pathogenesis is not yet understood. We sought to characterize DC-SIGN-binding glycoproteins in a panel of allergenic and non-allergenic foods. Fluorescent-labeled peanut and soy extracts were used to test protein binding to human monocyte-derived dendritic cells (DCs) by flow cytometry. DC-SIGN-blocking assays were performed by incubating DCs with food extracts followed by staining with anti-DC-SIGN antibody. Using a DC-SIGN-Fc chimera, food extracts were tested for binding by ELISA and autoradiography. IgE immunoblotting was performed with pooled sera from food-allergic subjects. DC activation and maturation were assessed by flow cytometry. We demonstrate that peanut agglutinin, a minor peanut allergen, is a novel ligand for DC-SIGN. Peanut agglutinin activates DCs to induce the expression of costimulatory molecules in vitro. We present a comprehensive report on the characterization of DC-SIGN-binding proteins in common allergenic foods such as peanut, soy, tree nuts, egg, and milk. Foods that rarely induce allergy, such as pine nuts, chickpea, and corn, showed no binding to DC-SIGN. Several DC-SIGN-binding proteins show reactivity in serum IgE immunoblots. We have also identified novel non-IgE-binding proteins that interact with DC-SIGN; these proteins may be important for regulating immune responses to these foods.

A Novel Multipeptide Microarray for the Specific and Sensitive Mapping of Linear IgE-Binding Epitopes of Food Allergens

Background: The identification of B-cell epitopes of food allergens can possibly lead to novel diagnostic tools and therapeutic reagents for food allergy. We sought to develop a flexible, low-tech, cost-effective and reproducible multipeptide microarray for the research environment to enable large-scale screening of IgE epitopes of food allergens. Methods: Overlapping peptides (15-mer, 4 amino acid offset) covering the primary sequence of either peanut allergen Ara h 1 or all 3 subunits of the soybean allergen Gly m 5 were simultaneously synthesized in-house on a porous cellulose matrix. Identical peptide microarrays created with up to 384 duplicate peptide-cellulose microspots each were investigated for specificity and sensitivity in IgE immunodetection and in direct experimental comparison to the formerly established SPOT™ membrane technique. Results: The in-house microarray identified with 98% reproducibility the same IgE-binding peptides as the SPOT™ membrane technique. Additional IgE-binding peptides were identified using the microarray. While the sensitivity was increased between 2- and 20-fold, the amount of human serum required was reduced by at least two thirds over the SPOT™ membrane technique using the microarray. After subtraction of the potential background, we did not observe non-specific binding to the presented peptides on microarray. Conclusions: The novel peptide microarray allows simple and cost-effective screening for potential epitopes of large allergenic legume seed storage proteins, and it could be adapted for other food allergens as well, to study allergenic epitopes at the individual subject level in large paediatric and adult study groups of food allergic subjects.

In vitro evaluation of intestinal epithelial TLR activation in preventing food allergic responses

Alterations in the gut microbiota composition are associated with food allergy. Toll-like receptors (TLRs) respond to microbial stimuli. We studied the effects of the ligation of TLRs on intestinal epithelial cells (IECs) in preventing an allergic effector response. IEC monolayers (T84 cells) were co-cultured with CD3/28-activated PBMCs from healthy controls or atopic patients and simultaneously apically exposed to TLR2, TLR4 or TLR9 ligands. The barrier integrity of T84 cell monolayers was significantly reduced upon co-culture with PBMCs of food allergic subjects compared to healthy subjects. Apical exposure of IECs to a TLR9 ligand prevented PBMC-induced epithelial barrier disruption. Using PBMCs from food allergic subjects, apical TLR9 activation on IECs increased the IFN-γ/IL-13 and IL-10/IL-13 ratio, while suppressing pro-inflammatory IL-6, IL-8 and TNF-α production in an IEC-dependent manner. Hence, the activation of apical TLR9 on IECs, potentially by microbiota-derived signals, may play an important role in the prevention of allergic inflammation.

Establishment of Reference Doses for residues of allergenic foods: Report of the VITAL Expert Panel

In 2011, an expert panel was assembled to establish appropriate Reference Doses for allergenic food residues as a part of the VITAL (Voluntary Incidental Trace Allergen Labeling) program of The Allergen Bureau of Australia & New Zealand (ABA). These Reference Doses would guide advisory labeling decisions for use on food labels. Individual NOAELs and LOAELs were obtained from clinical challenges of food-allergic subjects. Statistical dose-distribution models (log-normal, log–logistic, Weibull) were applied to the individual NOAELs and LOAELs for each allergenic food. The Reference Doses, in terms of mg of total protein from the allergenic food, were based upon either the ED01 (for peanut, cow’s milk), the 95% lower confidence interval of the ED05 (for wheat, soybean, cashew, shrimp, sesame seed, mustard, and lupine), or both (egg, hazelnut) using all appropriate statistical dose-distribution models. Reference Doses were established for 11 allergenic foods ranging from 0.03mg for egg protein to 10mg for shrimp protein. Reference Doses were not established for fish or celery due to poor model fits with existing data. Reference Doses were not established for other tree nuts beyond hazelnut and cashew because of the absence of data on NOAELs and LOAELs from individual subjects.

Altered T Helper 17 Responses in Children with Food Allergy

Background: While a central role for the T helper (Th) 1/Th2 axis in food allergy has been established, the Th17 response in food-allergic humans has not been addressed. Methods: Th17 responses in 18 peanut-allergic children, who were also allergic to at least one additional food allergen, were assessed relative to 15 age-matched healthy controls. To account for the atopy background in the allergic children, 7 atopic, but not food-allergic, individuals and their age-matched controls were included in this study. PBMCs were analyzed by flow cytometry ex vivo or were stimulated in vitro with peanut allergens, gliadin, or tetanus toxoid followed by analysis of proliferation and cytokine production in antigen-responsive cells. Results: We observed a significantly lower interleukin (IL) 17 production in CD4+ T cells of food-allergic individuals ex vivo (p < 0.02). In vitro, we found that IL-17 production in CD4+ T cells in response to all antigens tested was significantly impaired in food-allergic subjects compared to healthy controls (Ara: p < 0.005; gliadin: p < 0.004; TT: p < 0.03). No significant differences were observed between atopic and nonatopic individuals with no food allergy. Conclusion: Our results thus reveal a systemic, non-allergen-specific defect in Th17 responses to antigen stimulation in food allergic individuals, suggesting a role for Th17 cells in the control of food allergy and implicating IL-17 as a potential biomarker for tolerance to food antigens.

Identification and characterization of Lyc e 4, a Bet v 1 homologous PR-10 protein, in tomato fruits

Background Allergy to tomato (Lycopersicon esculentum) has been reported to occur in 3-16% of food allergic subjects. Tomato allergy is frequently manifested as OAS, urticaria and gastrointestinal symptoms. So far, three tomato allergens, Lyc e 1 (profilin), Lyc e 2 (invertase) and Lyc e 3 (nsLTP) are listed in the official IUIS allergen database. Although tomato allergy is frequently associated with birch pollen allergy in Central Europe, a cross-reactive Bet v 1 homologous allergen has not yet been described in tomato fruits. The objective of the present study was the cDNA cloning, recombinant expression and immunological characterization of Bet v 1 homologous PR10-proteins from tomato fruits.

Potential mechanisms for the association between fall birth and food allergy

Season of birth has been reported as a risk factor for food allergy, but the mechanisms by which it acts are unknown. Two populations were studied: 5862 children from the National Health and Nutrition Examination Survey (NHANES) III and 1514 well-characterized food allergic children from the Johns Hopkins Pediatric Allergy Clinic (JHPAC). Food allergy was defined as self-report of an acute reaction to a food (NHANES), or as milk, egg, and peanut allergy. Logistic regression compared fall or nonfall birth between (i) food allergic and nonallergic subjects in NHANES, adjusted for ethnicity, age, income, and sex, and (ii) JHPAC subjects and the general Maryland population. For NHANES, stratification by ethnicity and for JHPAC, eczema were examined. Fall birth was more common among food allergic subjects in both NHANES (OR, 1.91; 95% CI, 1.31-2.77) and JHPAC/Maryland (OR, 1.31; 95% CI, 1.18-1.47). Ethnicity interacted with season (OR, 2.34; 95% CI, 1.43-3.82 for Caucasians; OR, 1.19; 95% CI, 0.77-1.86 for non-Caucasians; P = 0.04 for interaction), as did eczema (OR, 1.47; 95% CI, 1.29-1.67 with eczema; OR, 1.00; 95% CI, 0.80-1.23 without eczema; P = 0.002 for interaction). Fall birth is associated with increased risk of food allergy, and this risk is greatest among those most likely to have seasonal variation in vitamin D during infancy (Caucasians) and those at risk for skin barrier dysfunction (subjects with a history of eczema), suggesting that vitamin D and the skin barrier may be implicated in seasonal associations with food allergy.

Altered Frequency And Composition Of Cultured Natural Killer Cells From Food Allergic Subjects

Altered Frequency And Composition Of Cultured Natural Killer Cells From Food Allergic Subjects

Identification of a Dau c PRPlike protein (Dau c 1.03) as a new allergenic isoform in carrots (cultivar Rodelika)

Up to 25% of food allergic subjects in central Europe suffer from carrot allergy. Until now, two isoforms of the major carrot (Daucus carota) allergen Dau c 1 have been described: Dau c 1.01, comprising five variants (Dau c 1.0101-Dau c 1.0105) and Dau c 1.02. To investigate potential allergenic properties of a Dau c PRPlike protein, a novel isoform of the PR-10 protein family in carrot. Dau c PRPlike cDNA from carrot roots (cv Rodelika) was cloned after RT-PCR and 5'RACE. Dau c PRPlike protein was expressed in E. coli, purified under native conditions by Ni-NTA chromatography and analysed by CD spectroscopy. Immuno-reactivity of the rDau c PRPlike protein was compared with rDau c 1.0104 and rDau c 1.0201 in terms of IgE binding (immunoblotting, ImmunoCAP), IgE cross-reactivity (ELISA inhibition) and in vitro mediator release with sera from carrot allergic patients. mRNA expression of Dau c PRPlike protein in wild-type and transgenic carrot roots was analysed by qRT-PCR. The Dau c PRPlike protein was identified as a new allergenic isoform, Dau c 1.03, in carrot roots. 68% of carrot allergic patients were sensitized to rDau c 1.03. The IgE-reactivity of rDau c 1.03 strongly correlated with reactivity to rDau c 1.0104, but not to rDau c 1.0201. The extent of IgE cross-reactivity and allergenic potency of Dau c 1 isoforms varied between the individual sera tested. Dau c 1.03 mRNA transcripts were up-regulated in Dau c 1.01 and Dau c 1.02 gene-silenced carrot roots. Dau c 1 isoforms display distinct IgE epitope heterogeneity. Dau c 1.03 appears to contribute to the allergenicity of carrots and the manifestation of carrot allergy. The epitope diversity of different Dau c 1 isoforms should be considered for component-resolved diagnosis and gene silencing of carrot allergens.

Experimental Food Allergy with defective formation of induced Regulatory T (iTreg) Cells: Phenotype Rescue With Allergen-Specific Treg cell therapy

RATIONALE: Mechanisms underlying the breakdown of oral tolerance in food allergic subjects are poorly understood. We hypothesized that a Th2-biased gut cytokine milieu subverts the tolerogenic iTreg cell response to food antigens into a pathogenic one. EXPERIMENTAL DESIGN: Mice with a gain of function mutation in the IL-4Ra immunotyrosine inhibitory motif (Il4raF709) and wild-type (WT) control mice were orally sensitized and challenged with Ovalbumin (OVA). Naïve T cells were converted into iTreg cells using TGF-beta in the absence or presence of graded concentrations of IL-4. iTreg cells bearing the OVA323-339 peptide-specific D011.10 T cell receptor were administered at the onset of the sensitization protocol in phenotype rescue experiments. RESULTS: . Il4raF709 but not WT mice were readily sensitized to orally administered OVA even in the absence of an adjuvant, and responded to subsequent oral challenge with OVA with IgE mediated anaphylaxis. OVA-stimulated mesenteric lymph node cultures exhibited decreased expansion of Foxp3+ T cells in OVA-sensitized Il4raF709 relative to WT mice. Purified naïve T cells of WT and Il4raF709 mice were equally converted into induced iTreg cells using TGF-beta. However, IL-4 disproportionately inhibited iTreg formation in Il4raF709 as compared to WT controls, resulting instead in heightened production by the TGF-beta treated cells of Th2 cytokines. In vivo, administration of OVA-specific iTreg cells at the onset of the sensitization protocol abrogated the induction of anaphylaxis in Il4raF709 mutant mice. CONCLUSION: Heightened IL-4R signaling may precipitate food allergy by antagonizing the formation of oral allergen-specific iTreg cells, promoting instead pathogenic Th2 cells.

Immunoglobulin E‐reactivity of wheat‐allergic subjects (baker's asthma, food allergy, wheat‐dependent, exercise‐induced anaphylaxis) to wheat protein fractions with different solubility and digestibility

Baker's asthma, food allergy to wheat, and wheat-dependent, exercise-induced anaphylaxis (WDEIA) are different clinical forms of wheat allergy. We investigated the correlation of solubility and digestion stability of wheat allergens with the IgE-reactivity patterns of different patient groups. Three wheat protein fractions were extracted according to their solubility: salt-soluble albumins and globulins, ethanol-soluble gliadins, and glutenins soluble only after treatment with detergents and reducing reagents. Sera from subjects with history of each variant of wheat allergy were characterized by CAP FEIA and immunoblotting. There was a high degree of heterogeneity of recognized allergens between the different subject groups as well as within these groups. However, subjects with WDEIA showed similar immunoglobulin E (IgE)-reactivity patterns to gliadins and especially to a 65 kDa protein. Subjects with baker's asthma as well as the food-allergic subjects had the most intense IgE-reactivity to the albumin/globulin fraction. The latter group additionally showed IgE-reactivity to the other fractions. Divergent results of immunoblotting and CAP-FEIA demonstrated that the detection of wheat-specific IgE highly depends on the applied method, thus the diagnostic tool must be carefully chosen. Most wheat allergens were rapidly digested as analyzed by determination of IgE-reactivity on immunoblots to wheat extracts after simulation of gastric and duodenal digestion. However, ethanol-soluble gliadins were stable to gastric enzymes and exhibit low solubility in gastric and duodenal fluids. Therefore, they are likely to be important in food allergy to wheat.

Murine IgE and IgG antibody responses to allergenic and non-allergenic foods: Potential model for allergenicity assessment of foods in man

Rationale Previous studies were directed at defining potential allergenicity of proteins using a mouse model. Intragastric (ig) immunizations of mice with peanut and shrimp extracts plus cholera toxin (CT) yielded IgE antibody responses to these allergens similar to the responses of food-allergic patients. Our current study includes additional results of mice immunized to a total of 5 major food allergens and 3 non-allergenic foods. Methods 10–20 C 3H/HeJ female mice, 7–8 weeks old, were immunized ig 4 times over a 6-week period with allergenic (shrimp, peanut, walnut, cashew, and cod) or non-allergenic (rice, chicken, and beef) food extracts plus 10 ug CT. Following exanguination, serum IgE and IgG antibody responses to foods were determined by ELISA. Results Significant IgE (0.7219 - 11.3 ug/ml) and IgG (172.2 - 1373 ug/ml) antibody responses to shrimp, peanut, walnut, and cashew were demonstrated. Minimal IgE antibody responses (0 - 0.0361 ug/ml) and lower IgG antibody responses (0.0331 - 461.7 ug/ml) were demonstrated to cod, rice, chicken, and beef. Immunoblotting showed that the IgE reactivity of food-allergic subjects and that of mice immunized to shrimp, peanuts, and nuts were similar. Conclusions These studies indicate that mice and man respond similarly to 4 out of 5 major food allergens and 3 out of 3 non-allergens, suggesting that the murine reaction to most major food allergens is similar to that of man. These results provide a basis for the future use of the mouse model in evaluating potential food allergens.

Carrot allergy: Double-blinded, placebo-controlled food challenge and identification of allergens

Background: Allergic reactions to carrot affect up to 25% of food-allergic subjects. Clinical manifestations of carrot allergy and IgE responses to carrot proteins, however, have never been studied in subjects with carrot allergy confirmed by means of double-blinded, placebo-controlled food challenge (DBPCFC). Objective: The purposes of this investigation were to confirm clinically relevant sensitizations to carrot by means of DBPCFC, to validate current diagnostic methods, and to identify IgE-reactive carrot proteins in patients with true allergy. Methods: DBPCFCs were performed in 26 subjects with histories of allergic reactions to carrot. Patients underwent skin prick tests with carrot extract, fresh carrot, and various pollen extracts. Specific IgE to carrot, celery, birch, and mugwort pollen and to rBet v 1, rBet v 2, and rBet v 6 were measured through use of the CAP method. Carrot allergens were identified by means of immunoblotting and blotting inhibition. Results: Twenty of 26 patients had positive DBPCFC results. The sensitivity of the determination of carrot-specific IgE antibodies through use of the CAP method (≥0.7 kU/L) was 90%, the sensitivity for skin prick testing with commercial extracts was 26%, and the sensitivity for prick-to-prick tests with raw carrot was 100%. The Bet v 1–related major carrot allergen Dau c 1 was recognized by IgE from 85% of patients; 45% were sensitized to cross-reactive carbohydrate determinants and 20% to carrot profilin. In 1 subject, a Bet v 6–related carrot allergen was recognized. In 4 patients, IgE binding to Dau c 1 was not inhibited or was weakly inhibited by rBet v 1 or birch pollen extract. Conclusion: This study confirmed the allergenicity of carrot by means of DBPCFC. DBPCFC-positive patients had exclusively specific IgE antibodies to birch pollen–related carrot allergens, Dau c 1 being the major allergen. The lack of inhibition of IgE binding to Dau c 1 by birch allergens in a subgroup of patients might indicate an secondary immune response to new epitopes on the food allergen that are not cross-reactive with Bet v 1. (J Allergy Clin Immunol 2001;108:301-7.)

Evidence for circulating IgE complexes in food allergy.

Antigen entry and the formation of immune complexes occur in atopic subjects after food ingestion. We have studied patients with asthma, eczema and arthralgia and we were able to detect the presence of IgE immune complexes and the antigen entry from the gut before and after food challenge. Food allergic subjects showed, after food challenge, the presence of IgE and IgG immune complexes, which correlates with the subsequent occurrence of symptoms. Both the symptoms and the appearance of IgE immune complexes could be prevented by pretreatment with oral sodium cromoglycate (SCG). IgE immune complexes were isolated by an isokinetic ultracentrifugation gradient and the components were analyzed for antigen-specific IgE and IgG as well as free antigen levels. The observation that SCG reduces antigen entry, the appearance of IgE immune complexes and the occurrence of symptoms point to the central role of IgE in the intestinal mucosa.