Effects of enzymatic hydrolysis combined with pressured heating on tree nut allergenicity.

Hazelnut, pistachio and cashew are tree nuts with health benefits but also with allergenic properties being prevalent food allergens in Europe. The allergic characteristics of these tree nuts after processing combining heat, pressure and enzymatic digestion were analyzed through in vitro (Western blot and ELISA) and in vivo test (Prick-Prick). In the analyzed population, the patients sensitized to Cor a 8 (nsLTP) were predominant over those sensitized against hazelnut seed storage proteins (Sprot, Cor a 9 and 14), which displayed higher IgE reactivity. The protease E5 effectively hydrolyzed proteins from hazelnut and pistachio, while E7 was efficient for cashew protein hydrolysis. When combined with pressured heating (autoclave and Controlled Instantaneous Depressurization (DIC)), these proteases notably reduced the allergenic reactivity. The combination of DIC treatment before enzymatic digestion resulted in the most effective methodology to drastically reduce or indeed eliminate the allergenic capacity of tree nuts.

Validation of the NUT CRACKER Diagnostic Algorithm and Prediction for Cashew and Pistachio Co-Allergy.

BACKGROUND: Because of the high cross-sensitization among tree nuts, the NUT CRACKER (Nut Co-reactivity-Acquiring Knowledge for Elimination Recommendations) study proposed a diagnostic algorithm to minimize the number of required oral food challenges (OFCs). OBJECTIVE: To validate the algorithm for cashew and pistachio allergy and determine markers for allergic severity. METHODS: Patients (n = 125) with a median age of 7.8 (interquartile range, 5.9-11.2) years with suspected tree nut allergy were evaluated prospectively with decision tree points on the basis of skin prick test (SPT), basophil activation test (BAT), and knowledge of the coincidence of allergies. Validation of allergic status was determined by OFC. Markers of clinical severity were evaluated using the combined original and prospective cohort (n = 187) in relationship to SPT, BAT, and Ana o 3-sIgE. RESULTS: Reactivity to cashew in SPT, BAT, and Ana o 3-sIgE and the incidence of abdominal pain on challenge were significantly higher in dual-allergic cashew/pistachio patients (n = 82) versus single cashew allergic patients (n = 18) (P = .001). All 3 diagnostic tests showed significant inverse correlation with log10 reaction doses for positive cashew OFC. The algorithm reduced overall the total number of OFCs by 72.0%, with a positive predictive value and negative predictive value of 93.0% and 99.0%, respectively. Cashew false-positives were observed primarily in hazelnut-allergic patients (P = .026). In this population, Ana o 3-specific IgE could diagnose cashew allergy with a sensitivity of more than 90% and a specificity of more than 95%. CONCLUSIONS: The NUT CRACKER diagnostic algorithm was validated and reduced the number of diagnostic OFCs required. Markers for severity phenotypes may guide oral immunotherapy protocols, improving the risk/benefit ratio for patients.

Cashew oral immunotherapy for desensitizing cashew-pistachio allergy (NUT CRACKER study).

BACKGROUND: Oral immunotherapy (OIT) is a treatment option for patients with milk, egg, and peanut allergy, but data on the efficacy and safety of cashew OIT are limited. METHODS: A cohort of 50 cashew-allergic patients aged ≥4 years, who were consecutively enrolled into cashew OIT (target dose 4000 mg protein) between 4/2016 and 12/2019. Fifteen cashew-allergic patients who continued cashew elimination served as observational controls. Co-allergy to pistachio and walnut was determined. Full desensitization rate and associated immunological changes in both groups were compared. Patients fully desensitized to cashew were instructed to consume a dose of 1200 mg cashew protein for 6 months and were then challenged to a full dose. Patients with co-allergy to pistachio or walnut were challenged to the respective nut. RESULTS: Forty-four of 50 OIT-treated patients (88%) compared to 0% in controls tolerated a dose of 4000 mg cashew protein at the end of the study (odds ratio 8.3, 95% CI 3.9-17.7, p < 0.001). An additional three patients were desensitized to 1200 mg cashew protein, and three patients stopped treatment. Three patients (6%) were treated with injectable epinephrine for home reactions. Desensitized patients had decreased SPT, sIgE, basophil reactivity, and increased sIgG4, following treatment. Following cashew desensitization, all pistachio (n = 35) and four of eight walnut co-allergic patients were cross-desensitized to the respective nut. All (n = 44) patients consuming a low cashew dose for ≥6 months following desensitization passed a full-dose cashew OFC. CONCLUSIONS: Cashew OIT desensitizes most cashew-allergic patients and cross-desensitizes to pistachio. Safety is similar to OIT for other foods.

Pistachio and cashew nut allergy in childhood: Predictive factors towards development of a decision tree.

BACKGROUND: Pistachio and cashew nut, which belong to the same botanical family, are tree nuts that induce serious allergic reactions. OBJECTIVE: We aimed to determine the predictive factors for pistachio and cashew nut reactivity during oral food challenge (OFC). METHODS: A total of 112 pistachio and/or cashew nut sensitized children, aged 58.45 (IQR:40.38-88.32) months, were included. Cutoff values and probability curves for skin prick test (SPT), sIgE, sIgE/Total IgE that predict reactivity were determined for pistachio and cashew nut. Additionally, a diagram was created that can be useful while making a decision for OFC based on SPT and sIgE values. RESULTS: A total of 73 patients underwent OFC with pistachio and/or cashew nut. Twelve children with current anaphylaxis history were not challenged and accepted as allergic. SPT was the only predictive factor for positive pistachio/ cashew nut OFC. According to area under curve (AUC) analysis, SPT was more predictive than sIgE and sIgE/Total IgE both for pistachio and cashew nut. Optimal cutoff values according to "Youden index" for pistachio SPT, sIgE, and sIgE/ Total IgE were 7.25 mm, 4.14 kUA/L, and 1.32%, respectively. And those values for cashew nut SPT, sIgE, and sIgE/Total IgE were 6.25 mm, 1.125 kUA/L, and 3.30%, respectively. The diagram showed that SPT predicted the reactivity together with sIgE better than only the SPT values. CONCLUSION: SPT was the best predictor for reactivity both for pistachio and cashew nut. Combined use of SPT and sIgE may improve the prediction of reactivity at pistachio and cashew nut OFCs in children.

Immunoglobulin E-Binding Pattern of Canadian Peanut Allergic Children and Cross-Reactivity with Almond, Hazelnut and Pistachio.

Peanut allergic individuals can be both co-sensitized and co-allergic to peanut and tree nuts. At the moment, standard diagnostic approaches do not always allow differentiation between clinically relevant sensitization and nonsignificant cross-reactions, and the responsibility of each allergen remains unclear. The objective of this study was therefore to determine a peanut sensitization profile in a cohort of Canadian peanut allergic children and assess the immunoglobulin E (IgE) molecular cross-reactivity between peanut, almond, hazelnut and pistachio. The specific IgE (sIgE) levels of each patient serum were determined by ImmunoCAP, indirect ELISA and immunoblot to examine their sIgE-binding levels and profiles to peanut proteins. Reciprocal inhibition ELISA and immunoblotting were used to study sIgE cross-reactions between peanut and the selected tree nuts using an adjusted and representative serum pool of the nine allergic patients. The results showed that the prepared peanut and tree nut protein extracts allowed for the detection of the majority of peanut and selected tree nut known allergens. The reciprocal inhibition ELISA experiments showed limited sIgE cross-reactivities between peanut and the studied tree nuts, with peanut being most likely the sensitizing allergen and tree nuts the cross-reactive ones. In the case of hazelnut and pistachio, a coexisting primary sensitization to hazelnut and pistachio was also demonstrated in the serum pool. Reciprocal inhibition immunoblotting further revealed that storage proteins (2S albumin, 7S vicilin and 11S legumin) could possibly account for the observed IgE-cross-reactions between peanut and the studied tree nuts in this cohort of allergic individuals. It also demonstrated the importance of conformational epitopes in the exhibited cross-reactions.

Predictive factors of allergy to pistachio in children allergic to cashew nut.

BACKGROUND: Cashew nut (CN) allergy prevalence has increased over the last few years. In children allergic to CN, complete avoidance of pistachio is usually recommended, but recent study showed that only one third of children allergic to CN were also allergic to pistachio. The aim of our study was to identify predictive factors of allergy to pistachio in children allergic to CN. METHODS: All children who had a positive oral food challenge (OFC) to CN between November 2013 and October 2017 in the Paediatric Allergy Department of the University Hospital of Nancy were included. Logistic regression models were used to predict the probability of allergy to pistachio. RESULTS: Among the 147 children included, tolerance or allergy to pistachio was known for 51. Out of these, 40 were allergic to pistachio (78.4%). Children allergic to pistachio had a larger weal size of skin prick test to CN (P = .01) and pistachio (P = .0007) and a lower reaction dose to CN (P < .0001). In multivariate analysis, only the reaction dose to CN was significantly associated with allergy to pistachio. Children with a low reaction dose to CN were significantly more at risk to have an allergy to pistachio (P = .01). CONCLUSION: A low reaction dose to CN seems to be a predictive factor of allergy to pistachio in children allergic to CN. In order to limit unnecessary food eviction, a pistachio OFC should be performed in children having high reaction dose whatever the importance of the skin or the specific IgE sensitization to pistachio.

Pistachio nut allergy: An updated overview.

Pistachio nut (Pistacia vera) is highly appreciated for its organoleptic characteristics and potential health benefits. However, this tree nut is also responsible for triggering moderate to severe IgE-mediated reactions in allergic individuals. Currently, pistachio nut allergy has gained some special attention, mainly due to its intrinsic relation with cashew nut allergy. Like for other nuts, the prevalence of pistachio nut allergy seems to be increasing at a global scale. Until now, there are five allergenic proteins officially listed for pistachio nut (Pis v 1, Pis v 2, Pis v 3, Pis v 4 and Pis v 5). Relevant data on their biochemical classification has become available, enabling establishing a correlation with the respective clinical symptoms. The establishment of an effective allergen risk assessment is a key issue for the food industry, policy makers and regulatory agencies. Thus, the availability of fast, specific and sensitive methods to detect trace amounts of allergens in processed foods is crucial. In the specific case of pistachio nut, there are some protein- and DNA-based methods for its detection/quantification in foods, which can aid to verify label information. Accordingly, all relevant research advances on this topic were summarised, updated and critically discussed in this review.

Thermal processing effects on the IgE-reactivity of cashew and pistachio.

Thermal processing can modify the structure and function of food proteins and may alter their allergenicity. This work aimed to elucidate the influence of moist thermal treatments on the IgE-reactivity of cashew and pistachio. IgE-western blot and IgE-ELISA were complemented by Skin Prick Testing (SPT) and mediator release assay to determine the IgE cross-linking capability of treated and untreated samples. Moist thermal processing diminished the IgE-binding properties of both nuts, especially after heat/pressure treatment. The wheal size in SPT was importantly reduced after application of thermally-treated samples. For cashew, heat/pressure treated-samples still retain some capacity to cross-link IgE and degranulate basophils, however, this capacity was diminished when compared with untreated cashew. For pistachio, the degranulation of basophils after challenge with the harshest heat/pressure treatment was highly decreased. Boiling produced more variable results, however this treatment applied to both nuts for 60 min, led to an important decrease of basophil degranulation.

Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

High-resolution Orbitrap™-based mass spectrometry for rapid detection of peanuts in nuts.

Peanut represents one of the most harmful allergenic foods capable of triggering severe and sometimes lethal reactions in allergic consumers upon ingestion of even small amounts. Several proteins capable of inducing allergic reactions that have been recognised by patients' IgE antibodies have been identified from this nut source. Methods mainly based on ELISA assays have been developed in order to detect peanuts in several food commodities. In addition LC-MS/MS methods based on different mass analysers have also been devised for tracing peanut contamination in different foods achieving low limits of detection. The applicability of a benchtop high-resolution Exactive™ mass spectrometer has never been investigated for the rapid screening of peanut contamination in complex food matrices like mixtures of nuts. We report in this paper the design of suitable peanut markers and the development of an high-resolution Orbitrap™ mass spectrometer-based method for peanut detection in a mixture of nuts species. With this aim, different types of samples were prepared: (1) nuts-based powder made up of a mixture of hazelnuts, pistachios, almonds and walnuts; and (2) nuts powder fortified with peanuts. Different levels of fortifications were produced and the applicability of the method was tested. Finally, a subset of six peptides fulfilling specific analytical requirements was chosen to check the suitability of the method tailored to the detection of peanuts in nuts-based products, and two of them, peptides VYD and WLG, were selected as quantitative markers. The method proved to be a suitable screening tool to assess the presence of traces of peanuts in other tree nuts with a limit of detection as low as 4 µg of peanuts proteins or 26 µg of peanuts in 1 g of matrix.

Pistachio allergy-prevalence and in vitro cross-reactivity with other nuts.

BACKGROUND: Tree nut allergy is characterized by a high frequency of life-threatening reactions and is typically lifelong persistent. Some people with a pistachio nut allergy, which is common in the pistachio rich area of Iran, develop a hypersensitivity to other tree nuts as well. The aim of this study was to investigate the prevalence of pistachio nut allergy in Iran, the major pistachio cultivation region in the world. The study also addressed the presence of allergenic cross-reactivity between pistachio and other nuts, including almond, peanut, and cashew in pistachio allergic patients. METHODS: A survey was conducted to determine whether the prevalence of pistachio allergy is affected by exposure to this nut in pistachio cultivation regions, as well as possible cross-reactivity between pistachio and other nuts including cashew, almond, and peanut. Inhibition Western blot and inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between pistachio and the other tree nuts. RESULTS: Our results revealed that the prevalence of pistachio allergy is twice as much in pistachio cultivation regions than other areas. Western blotting and inhibition ELISA presented high percentages of inhibition with pistachio and cashew, followed by almond and, to some degree, peanut which indicates different levels of allergenic cross-reactivity. CONCLUSIONS: The results indicate that exposure of people to pistachio significantly affects the prevalence of its allergic reactions. In addition, it was observed that, among pistachio allergic subjects, such exposure may affect the co-sensitivities with other nuts, including cashew and almond. The plant taxonomic classification of pistachio and other tree nuts does appear to predict allergenic cross-reactivity.

Single-tree nut immunotherapy attenuates allergic reactions in mice with hypersensitivity to multiple tree nuts.

BACKGROUND: Allergic reactions to tree nuts are often severe and are outgrown in less than 10% of diagnosed patients. OBJECTIVES: To determine whether treatment of underlying tree nut sensitization will prevent allergic reactions to cross-reacting tree nuts and to determine the effects of single-tree nut immunotherapy on true multi-tree nut sensitization. METHODS: Cross-reactivity model: Cashew-sensitized mice underwent immunotherapy with cashew and were subsequently challenged with cashew and pistachio. Multisensitization model: Cashew plus walnut-sensitized mice were treated with cashew alone, walnut alone, or both cashew and walnut and then underwent challenges to cashew and walnut. Challenges were assessed on the basis of symptoms, changes in body temperature, and mouse mast cell protease-1 release. RESULTS: In the cross-reactivity model, cashew immunotherapy completely prevented allergic reactions on challenges with cashew or the cross-reactive pistachio. In the multisensitization model, mice with cashew plus walnut allergy were significantly protected from anaphylactic reactions on cashew challenge in both the cashew-alone and walnut-alone immunotherapy groups. Results from the walnut challenge demonstrated significantly decreased allergic responses in the walnut immunotherapy group, whereas mice in the cashew immunotherapy group experienced significantly lower symptoms. In the cross-reactivity model, immunotherapy effectively decreased IL-4 and IL-5 production and increased IL-12 relative to placebo while also inducing a 5-fold increase in specific IgG(1). CONCLUSION: Single-tree nut immunotherapy can effectively decrease allergic responses in both the cross-reactivity and multisensitization mouse models. Further studies are needed to determine which single-tree nut immunotherapies will be most effective for specific multi-tree nut allergy profiles.

Influence of processing on the allergenic properties of pistachio nut assessed in vitro.

Pistachio (Pistacia vera) is a tree nut that has been reported to cause IgE-mediated allergic reactions. This study was undertaken to investigate the distinctions between different cultivars of pistachio nut and the influence of different processing on the IgE-binding capacity of whole pistachio protein extracts. The influence of different processes on allergenicity was investigated using competitive inhibition ELISA and Western blotting assays. The Western blotting results of extracts from pistachio cultivars showed no marked difference among them. The IgE-binding capacity was significantly lower for the protein extract prepared from steam-roasted than from raw and dry-roasted pistachio nuts. The results of sensory evaluation analysis and hedonic rating proved no significant differences in color, taste, flavor, and overall quality of raw, roasted, and steam-roasted pistachio nut treatments. The most significant finding of the present study was the successful reduction of IgE-binding by pistachio extracts using steam-roast processing without any significant changes in sensory quality of product.

Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family.

BACKGROUND: IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. OBJECTIVE: We attempted to identify pistachio allergens and to clone the encoding genes. METHODS: Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. RESULTS: Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. CONCLUSION: Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified.

Clinical features of four cases with cashew nut allergy and cross-reactivity between cashew nut and pistachio.

BACKGROUND: Few cases of cashew nut (CN) allergy have been reported in Japan. We evaluated the clinical features of 4 cases with CN allergy and investigated the allergens involved. METHODS: In order to investigate the cross-reactivity between CN and pistachios, we performed ImmunoCAP inhibition tests using sera of 4 cases with positive histories of CN allergy and positive results of specific IgE measurement (ImmunoCAP) and skin prick tests. Furthermore, we analyzed the molecular weights of allergens of CN and pistachios by IgE-immunoblotting. RESULTS: Of the 4 cases (male : female = 1:3), there were 3 cases (patient #2-4) and 1 case (patient #1) of anaphylaxis and oral allergy syndrome, respectively. The initial symptom was an oropharyngeal symptom in 3 of the 4 cases, of which 2 cases developed anaphylaxis within 10 minutes after eating only a few pieces of CN. All 4 cases reacted positively to the skin prick test with CN, although 1 case of anaphylaxis tested negatively for CN by ImmunoCAP. Additionally, in 2 cases, IgE-binding to CN and pistachio were inhibited with both pistachios and CN, indicating cross-reactivity between CN and pistachios. IgE-immunoblotting of CN using sera from the 4 cases revealed 2 bands at molecular weights of approximately 33 kd and 42 kd, whereas that of pistachios showed a single band at 36 kd. However, IgE in all 4 sera did not bind to rAna o 2. CONCLUSIONS: In CN allergy, a small amount of CN could induce a severe anaphylactic reaction. Moreover, in cases of suspected CN allergy, reactions to not only CN but also pistachio, which could be cross-reactive to CN, should be examined.

Pistachio vicilin, Pis v 3, is immunoglobulin E-reactive and cross-reacts with the homologous cashew allergen, Ana o 1.

BACKGROUND: Patients allergic to cashew nuts often report allergy to pistachio, which could be a result of cross-reactivity between the two as both are members of the Anacardiaceae family. OBJECTIVE: Because cashew 7S globulin (vicilin, Ana o 1) is a recognized major allergen, we cloned the pistachio homologue and assayed it for IgE reactivity and cross-reactivity with Ana o 1. METHODS: Degenerate primers for 7S globulin were used in PCR to amplify DNA from a pistachio cDNA library. An isolate was sequenced, cloned and expressed in Escherichia coli. Reactivity to the allergen was screened by dot blot using 19 pistachio and/or cashew-allergic patients' sera. Cross-reactivity was investigated by inhibition dot- and Western immunoblot assays using pistachio/cashew-allergic patients' sera, and monoclonal antibodies (MAbs) raised against recombinant Ana o 1 (rAna o 1). RESULTS: An isolate was found that coded for a 7S vicilin-like protein, designated Pis v 3. IgE reactivity to Pis v 3 was found in the serum of seven of the 19 (37%) patients with histories of allergy to both pistachio and cashew or who were allergic to cashew but had never eaten pistachio. The seven patients with IgE that recognized rPis v 3 also recognized rAna o 1. Six of nine anti-rAna o 1 MAbs also showed reactivity to rPis v 3 on dot blots. CONCLUSION: Of the 37% of pistachio/cashew-allergic patients' sera that recognized the pistachio allergen, rPis v 3, all showed complete cross-reactivity with rAna o 1. The data does not identify the primary sensitizing agent but suggests that IgE reactivity to rPis v 3 and rAna o 1 is focused on the most conserved regions of the proteins. Clinical histories suggest that in some cases, cashew was the sensitizing agent. rPis v 3 is a likely contributor to the observed co-sensitivity to pistachio and cashew in some patients.

Identification of hidden allergens: detection of pistachio traces in mortadella.

An analytical method based on the detection of specific DNA was developed and applied to mortadella samples with and without pistachio (Pistacia vera). The method is proposed for the detection of traces of pistachio deriving from previous processes or from accidental contamination, since in predisposed individuals pistachios can cause allergic reactions leading to anaphylactic shock. Three pairs of primers were identified and tested by polymerase chain reaction (PCR) on mortadella samples prepared with pistachio. Accidental contamination was also simulated. The optimized PCR was able to detect the presence of pistachio, even at low concentrations. The primers pair PSTC 1-2 is suggested for unambiguous identification of pistachio in mortadella. The limit of detection for this primers pair was 100 mg kg-1. No interference was observed from other spices or ingredients utilized in the formulation of the mortadella. The method enabled the identification of possible traces of pistachio remaining in the production plant after less than thorough washing.