Extended boiling of peanut progressively reduces IgE allergenicity while retaining T cell reactivity.
BACKGROUND: Current peanut oral immunotherapy is hampered by frequent adverse events. It has been shown that boiling can reduce peanut allergenicity. Hypoallergenic peanut products have the potential to reduce treatment-related reactions during desensitisation. OBJECTIVE: To show that extended boiling (for up to 12 hours) can progressively reduce peanut allergenicity while retaining T-cell reactivity. METHODS: Raw peanuts were boiled for half, 1, 2, 4 and 12 hours in deionized water. After dehydration, boiled and raw peanuts were ground, defatted and soluble proteins extracted in PBS and cooking water (leachate) retained. SDS-PAGE, Western blot, inhibition ELISA, mass spectrometry and skin prick test were used to characterize changes to peanut allergens and human IgE-reactivity. T cell responses to raw and boiled peanut extracts were determined by proliferation of CD4+/CD25+/CD134+ T cells in peanut-allergic and non-allergic individuals. RESULTS: Extended boiling progressively reduced peanut allergenicity through a combination of leaching of allergens into cooking water, fragmentation of allergens, and denaturation of conformational epitopes. Two hour boiling led to an 8-fold reduction in IgE binding capacity of boiled peanuts as determined by inhibition ELISA, while 12 hour boiling led to a 19-fold reduction. Mass spectrometry revealed an increasing number of unique allergen peptides with longer boiling times. Raw, 2 hour and 12 hour boiled peanut extracts were equivalent in their ability to stimulate T cell activation and proliferation. CONCLUSION AND CLINICAL RELEVANCE: Progressive reduction of peanut allergenicity with extended boiling does not affect T cell reactivity. Boiled peanuts may be a candidate for oral immunotherapy.
Quantitative proteomic profiling of peanut allergens in food ingredients used for oral food challenges.
Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data independent acquisition coupled with ion mobility mass spec-trometry-mass spectrometry (DIA-IM-MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semi-quantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts, and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, whilst the Bet v 1 homologue Ara h 8 and the lipid transfer protein aller-gen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11 were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2 and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantifica-tion of peanut allergens which can be applied to both food ingredients used in clinical studies, extracts used for skin testing and to identify trace levels of allergens in foods.
The effect of thermal processing on the behaviour of peanut allergen peptide targets used in multiple reaction monitoring mass spectrometry experiments.
Mass spectrometry-based methods offer an alternative means of determining allergens in foods. Whilst targeted methods are likely to offer the most robust approach for detection and quantification, little is known about how food processing may affect the behaviour of peptide targets. A systematic study has been undertaken to investigate the effects of thermal processing (boiling, roasting, frying) on the behaviour of a suite of peanut peptide targets representing the major clinically-relevant allergens. Initially the effect of thermal processing on protein extractability was investigated and a mass spectrometry-compatible buffer identified comprising 50 mM Tris-HCl, pH 8.8 containing 50 mM dithiothreitol and 0.04% (w/v) acid labile detergent which was able to extract 45-100% of protein from raw, boiled, roasted and fried peanuts using sonication at 60 °C. Eight peptide targets were identified including two peptides from each cupin allergen, Ara h1 and Ara h3 and four peptides from the prolamin superfamily allergens Ara h2, 6 and 7. AQUA peptide standards were synthesised and used to undertake multiple-reaction monitoring experiments, giving assay sensitivities of 0.1-30 amoles of peptide on-column (3 : 1 signal : noise), calculated limits of quantification between 96-1343 amoles of peptide on-column and a linear dynamic range of 4-5 orders of magnitude. Absolute quantification of individual peanut allergens in thermally processed samples showed that peptide targets in the cupin allergens were more prone to processing-induced effects than those from Ara h2, 6 and 7. Targets flanked by arginine residues showed greater thermostability. Identification of processing-stable targets, coupled with more efficient extraction procedures and a wide dynamic range, shows that targeted mass spectrometry methods have great potential as an additional method for quantifying peanut allergens in complex food matrices.
Identification and characterization of a new pecan [Carya illinoinensis (Wangenh.)
The 7S vicilin and 11S legumin seed storage globulins belong to the cupin protein superfamily and are major food allergens in many foods from the "big eight" food allergen groups. Here, for the first time, pecan vicilin was found to be a food allergen. Western blot experiments revealed that 30% of 27 sera used in this study and 24% of the sera from 25 patients with double-blind, placebo controlled clinical pecan allergy contained IgE antibodies specific to pecan vicilin. This allergen consists a low-complexity region at its N-terminal and a structured domain at the C-terminal that contains two cupin motifs and forms homotrimers. The crystal structure of recombinant pecan vicilin was determined. The refined structure gave R/Rfree values of 0.218/0.262 for all data to 2.65 Å. There were two trimeric biological units in the crystallographic asymmetric unit. Pecan vicilin is also a copper protein. These data may facilitate the understanding of the nutritional value and the allergenicity relevance of the copper binding property of seed storage proteins in tree nuts.
Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts.
BACKGROUND: Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T-cell epitopes and T-cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. OBJECTIVES: In this study, we characterized cashew specific T-cell responses in cashew allergic subjects and examined cross-reactivity of these cashew specific cells toward other tree nut allergens. METHODS: CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T-cells was determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. RESULTS: CD4+ T-cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T-cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T-cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. CONCLUSIONS: Phylogenetically diverse tree nut allergens can activate cashew reactive T-cells and elicit a TH 2 type response at an epitope specific level.
Peanut allergy in Mexican children: what is the effect of age at first consumption?
Studies suggest that children who start solid foods early are at risk for developing food allergies. Herein, we evaluated the effects of the introduction of peanuts to the diets of children on emerging peanut allergies. Children with allergic rhinitis and asthma were enrolled in the present study and evaluated in four stages. In the first stage, a clinical history was completed for all participants. In the second stage, skin tests were conducted to detect the sensitization to peanuts. In the third stage, the parents were interviewed about the peanut-eating habits of their children. In the fourth stage, children with a convincing history of allergy or a positive peanut skin test result were subjected to an open oral food challenge (OOFC). Three hundred children in four groups were included, 58.2% of the subjects were male, and the mean age was 7.3±3.9 years. The median age of first exposure to peanuts in patients with peanut allergies was greater than that in children without peanut allergies (2 years versus 1 year; p=0.009). The multivariate analysis, including only those children subjected to the OOFC, revealed that the consumption of peanuts after the age of ≥2 years is a risk factor for developing a peanut allergy (odds ratio=8.0, 95% confidence interval 1.3-50.0, p=0.026). The results of the present study showed that the late introduction of peanuts to children increases the risk of developing a peanut allergy.
Effect of Avoidance on Peanut Allergy after Early Peanut Consumption.
Background In a randomized trial, the early introduction of peanuts in infants at high risk for allergy was shown to prevent peanut allergy. In this follow-up study, we investigated whether the rate of peanut allergy remained low after 12 months of peanut avoidance among participants who had consumed peanuts during the primary trial (peanut-consumption group), as compared with those who had avoided peanuts (peanut-avoidance group). Methods At the end of the primary trial, we instructed all the participants to avoid peanuts for 12 months. The primary outcome was the percentage of participants with peanut allergy at the end of the 12-month period, when the participants were 72 months of age. Results We enrolled 556 of 628 eligible participants (88.5%) from the primary trial; 550 participants (98.9%) had complete primary-outcome data. The rate of adherence to avoidance in the follow-up study was high (90.4% in the peanut-avoidance group and 69.3% in the peanut-consumption group). Peanut allergy at 72 months was significantly more prevalent among participants in the peanut-avoidance group than among those in the peanut-consumption group (18.6% [52 of 280 participants] vs. 4.8% [13 of 270], P<0.001). Three new cases of allergy developed in each group, but after 12 months of avoidance there was no significant increase in the prevalence of allergy among participants in the consumption group (3.6% [10 of 274 participants] at 60 months and 4.8% [13 of 270] at 72 months, P=0.25). Fewer participants in the peanut-consumption group than in the peanut-avoidance group had high levels of Ara h2 (a component of peanut protein)-specific IgE and peanut-specific IgE; in addition, participants in the peanut-consumption group continued to have a higher level of peanut-specific IgG4 and a higher peanut-specific IgG4:IgE ratio. Conclusions Among children at high risk for allergy in whom peanuts had been introduced in the first year of life and continued until 5 years of age, a 12-month period of peanut avoidance was not associated with an increase in the prevalence of peanut allergy. Longer-term effects are not known. (Funded by the National Institute of Allergy and Infectious Diseases and others; LEAP-On ClinicalTrials.gov number, NCT01366846).
Ethnic differences in prevalence of cashew nut, pistachio nut and almond allergy.
Allergy to peanuts and tree nuts is one of the most serious food allergies affecting children in English-speaking westernised countries like the USA, UK and Australia. The prevalence of peanut allergy (PA) in these regions is as high as 1-3% and is possibly increasing whereas it appears to be uncommon in developing communities in Africa and Asia (1-3). In both these high and low prevalence regions however differences of peanut sensitisation among various ethnic groups in the same geographical areas have been reported, with higher prevalences in immigrant ethnic minorities in the developed communities (4,5).
The component-specific to total IgE ratios do not improve peanut and hazelnut allergy diagnoses.
BACKGROUND: Specific IgE measurement predicts the outcome of oral food challenges with considerable uncertainty when evaluating food allergy. OBJECTIVE: Our aim was to assess whether accounting for the ratio of component- or allergen-specific to total IgE can improve this prediction. METHODS: This multicenter study collected blood samples from children with suspected peanut or hazelnut allergy referred to allergy specialist clinics for food challenges. Specific IgE to peanuts, hazelnuts, and their components (Ara h 1, Ara h 2, Ara h 3, Ara h 8, Cor a 1, Cor a 8, Cor a 9, and Cor a 14) and total IgE levels were determined by using the ImmunoCAP-FEIA. Specific to total IgE ratios were compared with raw IgE levels in terms of discrimination and prediction. RESULTS: Eighty-eight (43%) of 207 children with suspected peanut allergy and 44 (31%) of 142 children with suspected hazelnut allergy had symptoms during food challenge. Discrimination was similar for raw and ratio measures: areas under the curve of 0.93 for Ara h 2-specific IgE versus 0.92 for the Ara h 2-specific/total IgE ratio and 0.89 for Cor a 14-specific IgE versus 0.87 for the Cor a 14-specific/total IgE ratio. The probability for a positive peanut challenge with 0.35 kU/L Ara h 2-specific IgE was 16% when the total IgE level was greater than 500 kU/L compared with 51%/48% for low/medium total IgE levels (<100/100-500 kU/L). A positive hazelnut challenge with 0.35 kU/L Cor a 14-specific IgE was estimated in 7% when total IgE levels were high compared with 34%/32% with low/medium total IgE levels. CONCLUSIONS: Raw Ara h 2- and Cor a 14-specific IgE levels were the best single predictors for pediatric peanut and hazelnut allergies, suggesting the omission of challenges at very high levels. Calculating ratio measures did not improve prediction in this population. However, estimation of individual probabilities for challenge outcomes could be supported by total IgE levels because high levels might indicate lower probabilities at a given component-specific IgE level.
Multicentre Double-Blind Placebo-Controlled Food Challenge Study in Children Sensitised to Cashew Nut.
BACKGROUND: Few studies with a limited number of patients have provided indications that cashew-allergic patients may experience severe allergic reactions to minimal amounts of cashew nut. The objectives of this multicentre study were to assess the clinical relevance of cashew nut sensitisation, to study the clinical reaction patterns in double-blind placebo-controlled food challenge tests and to establish the amount of cashew nuts that can elicit an allergic reaction. METHODS AND FINDINGS: A total of 179 children were included (median age 9.0 years; range 2-17 years) with cashew nut sensitisation and a clinical history of reactions to cashew nuts or unknown exposure. Sensitised children who could tolerate cashew nuts were excluded. The study included three clinical visits and a telephone consultation. During the first visit, the medical history was evaluated, physical examinations were conducted, blood samples were drawn and skin prick tests were performed. The children underwent a double-blind placebo-controlled food challenge test with cashew nut during the second and third visits. The study showed that 137 (76.5%) of the sensitised children suspected of allergy to cashew nut had a positive double-blind placebo-controlled food challenge test, with 46% (63) manifesting subjective symptoms to the lowest dose of 1 mg cashew nut protein and 11% (15) developing objective symptoms to the lowest dose. Children most frequently had gastro-intestinal symptoms, followed by oral allergy and skin symptoms. A total of 36% (49/137) of the children experienced an anaphylactic reaction and 6% (8/137) of the children were treated with epinephrine. CONCLUSION: This prospective study demonstrated a strikingly high percentage of clinical reactions to cashew nut in this third line population. Severe allergic reactions, including anaphylaxis requiring epinephrine, were observed. These reactions were to minimal amounts of cashew nut, demonstrated the high potency of this allergens.