Detection of Food Allergens by Taqman Real-Time PCR Methodology.
Real-time PCR (polymerase chain reaction) has shown to be a very effective technology for the detection of food allergens. The protocol described herein consists on a real-time PCR assay targeting the plant ITS (Internal Transcribed Spacer) region, using species-specific primers and hydrolysis probes (Taqman) dual labeled with a reporter fluorophore at the 5' end (6-carboxyfluorescein, FAM) and a quencher fluorophore at the 3' end (Blackberry, BBQ). The species-specific real-time PCR systems (primers/probe) described in this work allowed the detection of different nuts (peanut, hazelnut, pistachio, almond, cashew, macadamia, walnut and pecan), common allergens present in commercial food products, with a detection limit of 0.1 mg/kg.
Exploratory analysis of CD63 and CD203c expression in basophils from hazelnut sensitized and allergic individuals.
BACKGROUND: Sensitization to hazelnut (HN) is frequent and requires clarification to determine whether this sensitization is clinically relevant. The aim of this study was to investigate basophil activation profiles in HN-sensitized and allergic subjects. METHODS: Basophil activation was determined by flow cytometric analyses of CD63 and CD203c expression using several HN allergen concentrations. Depending on their clinical reaction pattern, an oral allergy symptom group (OAS, n = 20), a systemic reaction group (n = 12) and a sensitized group without clinical symptoms (n = 20) were identified. Additionally, 10 non-allergic and non-sensitized individuals served as controls. RESULTS: CD63 and CD203c expression differed between allergic (OAS and systemic group) and sensitized subjects. The HN concentration required to activate 30% of CD203c+ basophils [effective concentration (EC)30] was significantly higher in sensitized versus the allergic group (p = 0.0089). This was more pronounced when the basophil allergen threshold sensitivity (CD-sens) was calculated (CD63: p = 0.018; CD203c: p = 0.009). CONCLUSION: Our data indicate that the basophil activation test may provide information to better distinguish between sensitized and allergic subjects if several allergen concentrations are considered. CD203c expression displayed a better discrimination compared to CD63; therefore, its diagnostic value might be superior compared with CD63.
Streamlining the analytical workflow for multiplex MS/MS allergen detection in processed foods
Allergenic ingredients in pre-packaged foods are regulated by EU legislation mandating their inclusion on labels. In order to protect allergic consumers, sensitive analytical methods are required for detect allergen traces in different food products. As a follow-up to our previous investigations, an optimized, sensitive, label-free LC-MS/MS method for multiplex detection of five allergenic ingredients in a processed food matrix is proposed. A cookie base was chosen as a complex food matrix and home-made cookies incurred with whole egg, skimmed milk, soy flour, ground hazelnut and ground peanut were prepared at laboratory scale. In order to improve the analytical workflow both protein extraction and purification protocols were optimized and finally a sensitive streamlined SRM based analytical method for allergens detection in incurred cookies was devised. The effect of baking on the detection of selected markers was also investigated.
Advances in food allergy in 2015.
This review highlights research advances in food allergy that were published in the Journal in 2015. The world of food allergy research continues to rapidly accelerate, with increasing numbers of outstanding submissions to the Journal. In 2015, important studies on the epidemiology of food allergy were published, suggesting differential rates of food allergy in specific racial and ethnic groups. Even more importantly, studies were published identifying specific risk factors for the development of peanut allergy, as well as specific prevention strategies. We also saw new studies on the diagnosis of food allergy and potential approaches to the treatment of food allergy, as well as novel mechanistic studies helping to explain the immunologic correlates of food allergy and food desensitization.
Identification and Characterization of Ana o 3 Modifications on Arginine Residue-111 in Heated Cashew Nuts.
Raw and roasted cashew nut extracts were evaluated for protein modifications by mass-spectrometry. Independent modifications on the Arg-111 residue of Ana o 3 were observed in roasted but not raw cashew nuts. The mass changes of 72.0064 or 53.9529 Da are consistent with formation of carboxyethyl and hydroimidazolone modifications at the Arg-111 residue. These same modifications were observed in Ana o 3 purified from roasted but not raw cashew nuts, albeit at a relatively low occurrence. Circular dichroism indicated that Ana o 3 purified from raw and roasted cashew nuts had similar secondary structure, and dynamic light scattering analysis indicated there was no observable difference in particle size. The stability of Ana o 3 purified from raw and roasted cashew nuts to trypsin was similar in the absence of or following treatment with a reducing agent. Only minor differences in IgE binding to Ana o 3 were observed by ELISA among a cohort of cashew allergic patient sera.
Endurance Exercise Increases Intestinal Uptake of the Peanut Allergen Ara h 6 after Peanut Consumption in Humans.
Controlled studies on the effect of exercise on intestinal uptake of protein are scarce and underlying mechanisms largely unclear. We studied the uptake of the major allergen Ara h 6 following peanut consumption in an exercise model and compared this with changes in markers of intestinal permeability and integrity. Ten overnight-fasted healthy non-allergic men (n = 4) and women (n = 6) (23 ± 4 years) ingested 100 g of peanuts together with a lactulose/rhamnose (L/R) solution, followed by rest or by 60 min cycling at 70% of their maximal workload. Significantly higher, though variable, levels of Ara h 6 in serum were found during exercise compared to rest (Peak p = 0.03; area under the curve p = 0.006), with individual fold changes ranging from no increase to an increase of over 150-fold in the uptake of Ara h 6. Similarly, uptake of lactulose (2-18 fold change, p = 0.0009) and L/R ratios (0.4-7.9 fold change, p = 0.04) were significantly increased which indicates an increase in intestinal permeability. Intestinal permeability and uptake of Ara h 6 were strongly correlated (r = 0.77, p < 0.0001 for lactulose and Ara h 6). Endurance exercise after consumption may lead to increased paracellular intestinal uptake of food proteins.
Epicutaneous immunotherapy for the treatment of peanut allergy in children and young adults. Journal of Allergy and Clinical Immunology
BACKGROUND: Peanut allergy is common, life-threatening, and without therapeutic options. We evaluated peanut epicutaneous immunotherapy (EPIT) by using Viaskin Peanut for peanut allergy treatment. OBJECTIVE: We sought to evaluate the clinical, safety, and immunologic effects of EPIT for the treatment of peanut allergy. METHODS: In this multicenter, double-blind, randomized, placebo-controlled study, 74 participants with peanut allergy (ages 4-25 years) were treated with placebo (n = 25), Viaskin Peanut 100 μg (VP100; n = 24) or Viaskin Peanut 250 μg (VP250; n = 25; DBV Technologies, Montrouge, France). The primary outcome was treatment success after 52 weeks, which was defined as passing a 5044-mg protein oral food challenge or achieving a 10-fold or greater increase in successfully consumed dose from baseline to week 52. Adverse reactions and mechanistic changes were assessed. RESULTS: At week 52, treatment success was achieved in 3 (12%) placebo-treated participants, 11 (46%) VP100 participants, and 12 (48%) VP250 participants (P = .005 and P = .003, respectively, compared with placebo; VP100 vs VP250, P = .48). Median change in successfully consumed doses were 0, 43, and 130 mg of protein in the placebo, VP100, and VP250 groups, respectively (placebo vs VP100, P = .014; placebo vs VP250, P = .003). Treatment success was higher among younger children (P = 0.03; age, 4-11 vs >11 years). Overall, 14.4% of placebo doses and 79.8% of VP100 and VP250 doses resulted in reactions, predominantly local patch-site and mild reactions (P = .003). Increases in peanut-specific IgG4 levels and IgG4/IgE ratios were observed in peanut EPIT-treated participants, along with trends toward reduced basophil activation and peanut-specific TH2 cytokines. CONCLUSIONS: Peanut EPIT administration was safe and associated with a modest treatment response after 52 weeks, with the highest responses among younger children. This, when coupled with a high adherence and retention rate and significant changes in immune pathways, supports further investigation of this novel therapy.
Enhanced Prophylactic and Therapeutic Effects of Polylysine-Modified Ara h 2 DNA Vaccine in a Mouse Model of Peanut Allergy.
BACKGROUND: The prevalence of food allergy has been increasing, but treatment is very limited. DNA vaccination has been recognized as a promising method for the treatment of allergic diseases; however, poor immunogenicity has hindered its application. METHODS: BALB/c mice were intradermally injected with plasmid DNA encoding the peanut protein Ara h 2 (pAra h 2) or pAra h 2 pretreated with poly-L- lysine (PLL) before or after sensitization with Ara h 2 protein. Ara h 2-specific antibodies were measured by ELISA. CD207+ dendritic cells (DCs) and Treg cells in draining lymph nodes were analyzed by flow cytometry after DNA immunization, and cytokine production in splenocytes was also analyzed. RESULTS: In the prophylactic study, pretreatment with pAra h 2 or PLL-pAra h 2 resulted in lower levels of Ara h 2-specific IgG1, IgG2a, and IgE after sensitization with Ara h 2 protein, and mice in the PLL-pAra h 2 group had a significantly lower level of antibodies than those in the pAra h 2 group. In the treatment study, intradermal injection with pAra h 2 or PLL-pAra h 2 after Ara h 2 protein sensitization significantly decreased the level of Ara h 2-specific antibodies, and PLL- pAra h 2 had stronger effects than pAra h 2. There were increased numbers of CD207+ DCs and Treg cells in the mice receiving intradermal injection with PLL-pAra h 2, and splenocytes from PLL-pAra h 2-treated mice secreted increased levels of IFN-γ and IL-10. CONCLUSIONS: Modification of pAra h 2 with PLL improved its prophylactic and therapeutic effects in peanut-allergic mice.
Desensitization for Peanut Allergies in Children.
Immunotherapy for peanut allergy has been an exploding topic of study within the last few years. Sublingual, epicutaneous, and oral immunotherapy are being investigated and show promise in the treatment of peanut allergy. Oral immunotherapy has shown the most clinical benefit; however, sublingual and epicutaneous immunotherapy appear to have the most favorable safety profiles. Most studies to date suggest that only a minority of subjects achieve sustained unresponsiveness to peanut after discontinuation of immunotherapy. Recent efforts have been focused on identifying adjunct therapies, such as omalizumab, that may assist patients in achieving peanut desensitization more quickly and with greater success. Several underlying immunologic mechanisms, including a switch from IgE to IgG4 production and induction of T regulatory cells, have been studied although more research is needed to identify reliable biomarkers. This article will describe the immunotherapy approaches that are being investigated to induce peanut desensitization, and highlight the benefits and risks of these therapies that need to be considered before they are ready for routine clinical practice.
Threshold Dose Distribution in Walnut Allergy.
BACKGROUND: In food allergy, eliciting doses (EDs) of foods on a population level can improve risk management and labeling strategies for the food industry and regulatory authorities. Previously, data available for walnut were unsuitable to determine EDs. OBJECTIVE: The objective of this study was to determine EDs for walnut allergic adults and to compare with previously established threshold data for peanut and tree nuts. METHODS: Prospectively, adult subjects with a suspected walnut allergy underwent a low-dose double-blind, placebo-controlled food challenge. Individual no observed and lowest observed adverse effect levels were determined and log-normal, log-logistic, and Weibull models were fit to the data. Estimated ED values were calculated for the ED5, ED10, and ED50, the dose respectively predicted to provoke an allergic reaction in 5%, 10%, and 50% of the walnut allergic population. RESULTS: Fifty-seven subjects were challenged and 33 subjects were confirmed to be walnut allergic. Objective symptoms occurred in 20 of the positive challenges (61%). The cumulative EDs in the distribution models ranged from 3.1 to 4.1 mg for the ED05, from 10.6 to 14.6 mg walnut protein for the ED10, and from 590 to 625 mg of walnut protein for the ED50. CONCLUSIONS: Our data indicate that population EDs for walnut are slightly higher compared with those for peanut and hazelnut allergy. Currently available data indicate that the ED values for hazelnut could be used as a conservative temporary placeholder when implementing risk management strategies for other tree nuts where little or no food challenge data are available.