Characterization of the B-cell receptor repertoires in peanut allergic subjects undergoing oral immunotherapy.
B-cell receptors (BCRs) play a critical role in adaptive immunity as they generate highly diverse immunoglobulin repertoires to recognize a wide variety of antigens. To better understand immune responses, it is critically important to establish a quantitative and rapid method to analyze BCR repertoire comprehensively. Here, we developed "Bcrip", a novel approach to characterize BCR repertoire by sequencing millions of BCR cDNA using next-generation sequencer. Using this method and quantitative real-time PCR, we analyzed expression levels and repertoires of BCRs in a total of 17 peanut allergic subjects' peripheral blood samples before and after receiving oral immunotherapy (OIT) or placebo. By our methods, we successfully identified all of variable (V), joining (J), and constant (C) regions, in an average of 79.1% of total reads and 99.6% of these VJC-mapped reads contained the C region corresponding to the isotypes that we aimed to analyze. In the 17 peanut allergic subjects' peripheral blood samples, we observed an oligoclonal enrichment of certain immunoglobulin heavy chain alpha (IGHA) and IGH gamma (IGHG) clones (P = 0.034 and P = 0.027, respectively) in peanut allergic subjects after OIT. This newly developed BCR sequencing and analysis method can be applied to investigate B-cell repertoires in various research areas, including food allergies as well as autoimmune and infectious diseases.
A Targeted LC-MS/MS Method for the Simultaneous Detection and Quantitation of Egg, Milk, and Peanut Allergens in Sugar Cookies.
Food allergy is a growing public health concern, with many individuals reporting allergies to multiple food sources. Compliance with food labeling regulations and prevention of inadvertent cross-contact in manufacturing requires the use of reliable methods for the detection and quantitation of allergens in processed foods. In this work, a novel liquid chromatography-tandem mass spectrometry multiple-reaction monitoring method for multiallergen detection and quantitation of egg, milk, and peanut was developed and evaluated in an allergen-incurred baked sugar cookie matrix. A systematic evaluation of method parameters, including sample extraction, concentration, and digestion, were optimized for candidate allergen peptide markers. The optimized method enabled the reliable detection and quantitation of egg, milk, and peanut allergens in sugar cookies, with allergen concentrations as low as 5 ppm allergen-incurred ingredient.
Assessing Almond and Peanut Allergens Using Commercially Available Immunoanalytical Kits and LC-MS/MS: A Case Study.
With an ever-increasing allergic population and an emerging market for allergen-free foods, accurate detection of allergens in foods has never been more important. Although ELISA-based methods are the most widely used for detection of allergens in food, there is a need for the development of orthogonal approaches. A commercial ELISA detected a relatively high concentration of peanut and almond in an allergen-free product. However, another commercial ELISA declared a low peanut concentration and was negative for almond. Further testing using a commercial almond lateral-flow device confirmed the results from the second ELISA kit and demonstrated that the positive detection of almond was due to cross-reactivity. An MS method was used for final confirmation that the reported results were negative for both almond and peanut.
Highly Sensitive Matrix-Independent Quantification of Major Food Allergens Peanut and Soy by Competitive Real-Time PCR Targeting Mitochondrial DNA.
The development of two competitive real-time PCR assays for the quantitative detection of trace amounts of twomajor food allergens, peanutand soybean, is reported. In order to achieve very low detection levels for both allergens, we established PCR primers and probes targeting mitochondrial DNA sequences. We were able to demonstrate that this approach led to an increase in detection sensitivity in the range of at least 1 order of magnitude compared with published assays targeting nuclear DNA. Furthermore, we generated corresponding competitor molecules, which were used as internal standards to compete with matrix effects that are evident during DNA extraction and PCR amplification in heterogeneous analytical matrixes like food. According to the recently described competitive quantitative PCR method published by Holzhauser et al. (2014), we performed threshold calibration against milk powder spiked with 10 ppm peanut and soy. Matrix-independent quantitative determination of peanut and soy could be demonstrated for three different calibrated food matrix standards in a range between 1 and 100 ppm. The data presented indicate that both assay concepts are powerful analytical tools for the quantitative detection of trace amounts of peanut and soy in commercial food products.
EAACI Guidelines on Allergen Immunotherapy: IgE‐mediated Food Allergy.
Food allergy can result in considerable morbidity, impairment of quality of life, and healthcare expenditure. There is therefore interest in novel strategies for its treatment, particularly food allergen immunotherapy (FA-AIT) through the oral (OIT), sublingual (SLIT), or epicutaneous (EPIT) routes. This Guideline, prepared by the European Academy of Allergy and Clinical Immunology (EAACI) Task Force on Allergen Immunotherapy for IgE-mediated Food Allergy, aims to provide evidence-based recommendations for active treatment of IgE-mediated food allergy with FA-AIT. Immunotherapy relies on the delivery of gradually increasing doses of specific allergen to increase the threshold of reaction while on therapy (also known as desensitization) and ultimately to achieve post-discontinuation effectiveness (also known as tolerance or sustained unresponsiveness). Oral FA-AIT has most frequently been assessed: here, the allergen is either immediately swallowed (OIT) or held under the tongue for a period of time (SLIT). Overall, trials have found substantial benefit for patients undergoing either OIT or SLIT with respect to efficacy during treatment, particularly for cow's milk, hen's egg, and peanut allergies. A benefit post-discontinuation is also suggested, but not confirmed. Adverse events during FA-AIT have been frequently reported, but few subjects discontinue FA-AIT as a result of these. Taking into account the current evidence, FA-AIT should only be performed in research centers or in clinical centers with an extensive experience in FA-AIT. Patients and their families should be provided with information about the use of FA-AIT for IgE-mediated food allergy to allow them to make an informed decision about the therapy.
Integrative transcriptomic analysis reveals key drivers of acute peanut allergic reactions. Nature communications.
Mechanisms driving acute food allergic reactions have not been fully characterized. We profile the dynamic transcriptome of acute peanutallergic reactions using serial peripheral blood samples obtained from 19 children before, during, and after randomized, double-blind, placebo-controlled oral challenges to peanut. We identify genes with changes in expression triggered by peanut, but not placebo, during acute peanut allergic reactions. Network analysis reveals that these genes comprise coexpression networks for acute-phase response and pro-inflammatory processes. Key driver analysis identifies six genes (LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3) predicted to causally modulate the state of coregulated networks in response to peanut. Leukocyte deconvolution analysis identifies changes in neutrophil, naive CD4+ T cell, and macrophage populations during peanut challenge. Analyses in 21 additional peanut allergic subjects replicate major findings. These results highlight key genes, biological processes, and cell types that can be targeted for mechanistic study and therapeutic targeting of peanut allergy.
Detection of IgE reactivity to a handful of allergen molecules in early childhood predicts respiratory allergy in adolescence.
BACKGROUND: Sensitization in early childhood may precede respiratory allergy in adolescence. METHODS: IgE reactivity against 132 allergen molecules was evaluated using the MeDALL microarray in sera obtained from a random sample of 786 children at the age of 4, 8 and 16years in a population based birth cohort (BAMSE). Symptoms were analyzed by questionnaire at ages 4, 8 and 16years. Clinically and independent relevant allergen molecules accounting for ≥90% of IgE reactivities in sensitized individuals and at all time-points were identified as risk molecules and used to predict respiratory allergy. The data was replicated in the Manchester Asthma and Allergy Study (MAAS) birth cohort by studying IgE reactivity with the use of a commercial IgE microarray. Sera were obtained from children at the ages of 3, 5, 8 and 11years (N=248) and the outcome was studied at 11years. FINDINGS: In the BAMSE cohort 4 risk molecules could be identified, i.e.: Ara h 1 (peanut), Bet v 1 (birch), Fel d 1 (cat), Phl p 1 (grass). For MAAS the corresponding number of molecules was 5: Der p 1 (dust mite), Der f 2 (dust mite), Phl p 1 (grass), Phl p 5 (grass), Fel d 1 (cat). In BAMSE, early IgE reactivity to ≥3 of 4 allergen molecules at four years predicted incident and persistent asthma and/or rhinitis at 16years (87% and 95%, respectively). The corresponding proportions in the MAAS cohort at 16years were 100% and 100%, respectively, for IgE reactivity to ≥3 of 5 risk molecules. INTERPRETATIONS: IgE reactivity to a few allergen molecules early in life identifies children with a high risk of asthma and/or rhinitis at 16years. These findings will be of importance for developing preventive strategies for asthma and rhinitis in children.
Recent advancement to prevent the development of allergy and allergic diseases and therapeutic strategy in the perspective of barrier dysfunction.
Therapeutic strategy in late 20th century to prevent allergic diseases was derived from a conceptual framework of allergens elimination which was as same as that of coping with them after their onset. Manifold trials were implemented; however, most of them failed to verify the effectiveness of their preventive measures. Recent advancement of epidemiological studies and cutaneous biology revealed epidermal barrier dysfunction plays a major role of allergen sensitization and development of atopic dermatitis which ignites the inception of allergy march. For this decade, therapeutic strategy to prevent the development of food allergy has been confronted with a paradigm shift from avoidance and delayed introduction of allergenic foods based on the theoretical concept to early introduction of them based on the clinical and epidemiological evidences. Especially, prevention of peanut allergy and egg allergy has been established with the highest evidence verified by randomized controlled trials, although application in clinical practice should be done with attention. This paradigm shift concerning food allergy was also due to the discovery of cutaneous sensitization risk of food allergens for an infant with eczema revealed by prospective studies. Here we have recognized the increased importance of prevention of eczema/atopic dermatitis in infancy. Two randomized controlled trials using emollients showed successful results in prevention of atopic dermatitis in infancy; however, longer term safety and prognosis including allergy march should be pursued. To establish more fundamental strategy for prevention of the development of allergy, further studies clarifying the mechanisms of interaction between barrier dysfunction and microbial milieu are needed with macroscope to understand the relationship between allergic diseases and a diversity of environmental influences.
Simultaneous Analysis of Multiple Allergens in Food Products by LC-MS/MS.
There is currently no cure for food allergies, and sufferers can only rely on the correct labeling of foods toavoid allergens. Hence, it is important that analytical methods are sensitive and accurate enough to screen for the presence of multiple allergens in food products. In this study, we developed an LC-tandem MS method that is able to simultaneously screen or quantify the signature tryptic peptides of multiple allergen commodities. This method is capable of screening and identifying egg white, skim milk, peanut, soy, and tree nuts (almond, Brazil nut, cashew, hazelnut, pecan, pine nut, pistachio, and walnut) at a detection limit of 10 ppm in incurred bread and cookies. It was further tested for the quantitative analysis of whole-egg, whole-milk, peanut butter, and hazelnut commodities, which are incurred or spiked into selected food matrixes as defined in AOAC INTERNATIONAL Standard Method Performance Requirement (SMPR®) 2016.002. The method demonstrated excellent sensitivity with a Method quantitative limit of 3 ppm for whole egg and 10 ppm for the remaining three allergen commodities. It also demonstrated good recovery (60‒119%) and repeatability (RSDr <20%), with an analytical range of 10-1000 ppm for each allergen commodity and was able to meet the minimum performance requirements of the SMPR.
Undeclared Food Allergens and Gluten in Commercial Food Products Analyzed by ELISA.
Undeclared allergen(s) in commercial food products are responsible for many food recalls, as reported by regulatory agencies in various countries, including the United States. Correct allergen labeling practices are essential for the safety of food-allergic consumers. However, this practice may be hindered by the introduction of allergens all along the food supply chain, including unintentionally through cross-contact. To understand the pervasiveness of undeclared allergen(s) in commercial food products, the objective of this review is to summarize the prevalence of undeclared milk, egg, hazelnut, peanut, soy, and gluten as detected by ELISA from previously published surveys. The prevalence of undeclared allergen(s) in products with or without an advisory statement was also summarized and compared. As compiled by this review, there are some food categories that may be at higher risk for containing undeclared allergen(s). However, the data on prevalence and amount of allergen present may vary widely within any particular allergen or food category. Factors, such as food survey product selection, geography, awareness of allergen/gluten issues, and/or the choice of ELISA method, may be responsible for such differences.