Project description:MS/MS analysis of the peanut protein extract (PPE) confirmed the presence of the four major peanut allergens and identified the presence of Ara h 1 isotypes, Ara h 2 isotypes, Ara h 3 isotypes, Ara h 6 isotypes and Ara h 7 isotypes. Allergen-specific immunotherapy (IT) is emerging as a viable option for treatment of peanut allergy. Yet, prophylactic IT remains unexplored despite early introduction of peanut in infancy was shown to prevent allergy. There is a need to understand how allergens interact with the immune system depending on the route of administration, and how different dosages of allergen may protect from sensitisation and a clinical active allergy. Here we compared peanut allergen delivery via the oral, sublingual (SL), intragastric (IG) and subcutaneous (SC) routes for the prevention of peanut allergy in Brown Norway (BN) rats. BN rats were administered PBS or three different doses of PPE via either oral IT (OIT), SLIT, IGIT and SCIT followed by intraperitoneal (IP) injections of PPE to assess the protection from peanut sensitisation. The development of IgE and IgG1 responses to PPE and the major peanut allergens were evaluated by ELISAs. The clinical response to PPE was assessed by an ear swelling test (EST) and proliferation was assessed by stimulating splenocytes with PPE. Low and medium dose OIT (1 and 10 mg) and all doses of SCIT (1, 10, 100 µg) induced sensitisation to PPE, whereas high dose OIT (100 mg), SLIT (10, 100 or 1000 µg) or IGIT (1, 10 and 100 mg) did not. High dose OIT and SLIT as well as high and medium dose IGIT prevented sensitisation from the following IP injections of PPE and suppressed PPE-specific IgE levels in a dose-dependent manner. Hence, administration of peanut protein via different routes confers different risks for sensitisation and protection from peanut allergy development. Overall, the IgE levels toward the individual major peanut allergens followed the PPE-specific IgE levels. Collectively, this study showed that the preventive effect of allergen-specific IT is determined by the interplay between the specific site of PPE delivery for presentation to the immune system, and the allergen quantity, and that targeting and modulating tolerance mechanisms at specific mucosal sites may be a prophylactic strategy for prevention of peanut allergy.

Project description:Background: Most asthmatic patients have high serum levels of IgE directed against common environmental allergens such as house dust mite, animal danders and moulds. However the presence of specific IgE against individual allergens alone does not account for asthma. Many individuals are atopic but not asthmatic. Precise knowledge of the serum IgE specificity repertoire in asthmatic and non-asthmatic patients would substantially help in understanding the pathogenesis of the disease and at the same time facilitate the treatment and the implementation of preventive measures. Methods: We developed a microarray immunoassay containing 103 common allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals from families whose members had a documented history of asthma and atopy. The results were analyzed using k-means clustering to investigate whether IgE reactivity profiles correlated with asthma, disease severity and age of onset as well as with other atopic conditions such as rhinitis, conjunctivitis and eczema. We trained an artificial neural network (ANN) using the serum reactivity data to identify individuals as asthmatic and non-asthmatic. Results: Individual sera showed clear differences in the number and the combinations of allergens recognized as well as in the level of specific IgE. While the presence of specific IgE against single allergens correlated poorly with the pathological conditions examined k-means clustering analysis unraveled that a particular profile was significantly associated with asthma (p <1E-8). An ANN-based algorithm, calibrated with the profile reactivity data correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Conclusions: Our analysis demonstrates that asthma may be a higher-order phenomenon related to patterns of response and not attributable to single antibody reactions. This information sheds new light on the risk of developing the disease and can be readily utilized in combination with an ANN-based tool to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile. The study consisted of a total of 872 sera samples: 485 individuals were diagnosed with asthma, 342 were classified as non asthmatic, a remaining 45 were classified as unconfirmed asthma diagnosis. The serum IgE reactivity profiles were analyzed against 103 allergens representative of 11 distinct allergen classes. Quantification of bound IgE: The fluorescence signal was acquired using ProScanArray Express™ version 3.0 software. PMC reading values of individual spots were corrected against the internal negative control to identify signals above background. Duplicate measurements of individual allergens were utilized. The signal collected from the allergens was interpolated with an external calibration curve to obtain the IU/ml value (see supplementary file GSE20020_IU_ml values.txt), and translated into a Class Score by plotting the data in a standard reactivity scale (see Sample data tables). Class Score values: (CLASS 0 (less than 0.35 IU/ml); CLASS 1 (0.35-0.7 IU/ml); CLASS 2 (0.71-3.5 IU/ml); CLASS 3 (3.51-17.5 IU/ml); CLASS 4 (17.51-50 IU/ml); CLASS 5 (50.01-100 IU/ml).

Project description:Cultivated peanut (Arachis hypogaea. L) is one of the most important oil crops in the world. After flowering, the peanut plant forms aboveground pegs (gynophores) that penetrate the soil, giving rise to underground pods. This means of reproduction, referred to as geocarpy, distinguishes peanuts from most other plants. The molecular mechanism underlying geocarpic pod development in peanut is poorly understood. To gain insight into the mechanism of geocarpy, we extracted proteins from aerial gynophores, subterranean unswollen gynophores, and gynophores that had just started to swell into pods. We analyzed the protein profiles in each of these samples by combining 1 DE with nanoLC-MS/MS approaches. In total, 2766, 2518, and 2280 proteins were identified from the three samples, respectively. An integrated analysis of proteome and transcriptome data revealed specifically or differentially expressed genes in the different developmental stages at both the mRNA and protein levels. A total of 69 proteins involved in the gravity response, light and mechanical stimulus, hormone biosynthesis, and transport were identified as being involved in geocarpy. Furthermore, we identified 91 genes that were specifically or abundantly expressed in aerial gynophores, including pectin methylesterase and expansin, which were presumed to promote the elongation of aerial gynophores. In addition, we identified 35 proteins involved in metabolism, defense, hormone biosynthesis and signal transduction, nitrogen fixation, and transport that accumulated in subterranean unswellen gynophores. Furthermore, 26 specific or highly abundant proteins related to fatty acid metabolism, starch synthesis, and lignin synthesis were identified in the early swelling pods. We identified thousands of proteins in the aerial gynophores, subterranean gynophores, and early swelling pods of peanut. This study provides the basis for examining the molecular mechanisms underlying peanut geocarpy pod development.

Dataset Information

Determination of allergen levels, isoforms and their hydroxyproline modifications among peanut genotypes by mass spectrometry

Publications

Determination of Allergen Levels, Isoforms, and Their Hydroxyproline Modifications Among Peanut Genotypes by Mass Spectrometry.

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