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Pathogenesis-related-10 (PR10) proteins play significant roles in plant defence against biotic and abiotic stresses. Recently, two banana PR10 proteins (MaPR10-BeB5 and MaPR10-GNA5) were characterised and shown to exhibit antifungal properties against Aspergillus fumigatus in vitro . In rice, transgenic overexpression of PR10 proteins conferred resistance to pathogen infection and drought tolerance without affecting productivity, highlighting their potential for agricultural applications. However, PR10 proteins also include the Bet v 1-like family of allergens implicated in pollen food allergy syndromes, raising concerns about potential adverse effects on human health. In this study, we evaluated the allergenic potential of the recently isolated banana PR10 proteins. We first predicted the presence of IgE epitopes of the Bet v 1 allergen family in the deduced PR10 peptide sequences in silico . We then predicted the structures of four human IgE scFv protein sequences and three plant PR10 protein sequences. Based on the quality of the predicted structures, one IgE scFv protein structure was selected for docking with the three plant PR10 proteins. We confirmed the docking results with immunoblot analysis performed using recombinant MaPR10-BeB5 and MaPR10-GNA5 proteins against the sera of banana-allergic patients. Our experimental results substantiated the notion that both protein variants are potentially allergenic since these proteins were recognised by 26.6% of banana-allergic patients with broad PR10 protein recognition. We caution that the allergenic potential of MaPR10 proteins should be carefully considered before implementing transgenic overexpression strategies to improve crops, with a suggestion to limit their expression to non-edible plant tissues.

Pru p 7 (also named Peamaclein) is a member of the gibberellin-regulated protein family, which is the latest foodborne allergenic protein identified in peach. In this paper, the prokaryotic expression and identification of Pru p 7 were performed, and the protein properties, structure, and homology were analyzed. In addition, a preliminary screening of B-cell linear epitopes of Pru p 7 was performed by the bioinformatics software prediction method, and three epitopes were identified using slot-blot immune microarray assay combined with an immune score matrix (P-1, AA16-21, AGYQER; P-2, AA40-46, TYGNKDE; P-3, AA52-59, DLKNSKGN). Moreover, the electrostatic potential of these epitopes was analyzed, and the stability after ultrahigh pressure treatment was also verified. Finally, the amino acids that play key immune roles in the epitopes were obtained by amino acid mutations. These results may contribute to the further understanding of Pru p 7 and the prevention of peach allergy.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli . In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis., Competing Interests: Conflicts of interest The authors have no conflict of interest to declare. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by USDA. USDA is an equal opportunity provider and employer., (Published by Elsevier Ltd.)

In this work, three allergen-encoding genes (Ana o 1, Ana o 2, Ana o 3) were investigated for the detection of cashew nut as an allergenic food. Normalised and single-tube nested real-time PCR approaches targeting the Ana o 2 or Ana o 3 genes are proposed and compared. Normalised real-time PCR detected 10 pg, while single-tube nested real-time PCR achieved 1 pg of cashew nut DNA. Single-tube nested real-time PCR targeting Ana o 3 allowed the best relative sensitivities (10 mg/kg cashew nut in dough/biscuit), being successfully validated regarding precision/accuracy. The normalised real-time PCR did not show acceptable accuracy for both targets. Sensitivity of single-tube nested real-time PCR was affected by the matrix (pasta), but not by thermal processing (dough/biscuit). Herein, two highly sensitive and specific single-tube nested real-time PCR targeting allergen-encoding genes are proposed for the first time as quantitative/validated tools for cashew nut analysis as an allergenic food., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Birch belongs to the order Fagales and the family Betulaceae. Birch pollen is one of the most important airborne inhaled allergens in the north temperate zone, leading to allergic rhinitis, asthma and pollen-related food allergy. The sensitization rate to birch pollen is about 8-16% in the general population and 7-57% in patients seen at various allergy centers. Seven birch pollen allergens have been recognized by the International Allergen Nomenclature Sub-committee, with Bet v 1 as the sole major allergen. Component-resolved diagnostics can help to discriminate broad cross-reactivity and false-positive diagnoses of pollen allergy caused by specific IgE to pan-allergens such as Bet v 2, 4 or Bet v 7 from true birch allergy represented by the major allergen Bet v 1-specific IgE. Patients with allergic symptoms to birch pollen showed significantly higher serum anti-Bet v 1 IgE concentrations than asymptomatic individuals with birch sensitization. A higher level of IgE to Bet v 1 also predicted oral allergy syndrome after the ingestion of Rosaceae fruits, nuts, or Apiaceae vegetables, which have cross-reactive homologous allergens with birch allergens. Bet v 1 is one of the first allergens developed using recombinant technology. Many forms of genetically modified Bet v 1 hypo-allergens have been developed and have shown benefit in animal models or even clinical trials of allergen immunotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Scope: Around 25% of food allergic persons in Central Europe suffer from carrot allergy caused by the major carrot allergen Dau c 1. Three different isoallergens, Dau c 1.01, Dau c 1.02 and Dau c 1.03 are identified. However, information about the qualitative and quantitative composition of natural (n)Dau c 1 is scarce., Methods and Results: The new carrot allergen Dau c 1.0401 is identified on the mRNA and protein level by RT-PCR and mass spectrometry. It displays only around 60% sequence identity to the other known Dau c 1 isoallergens. NMR and CD-spectra are typical for a well-folded protein containing both α-helices and β-strands. It showed a poor refolding capacity after incubation at 95 °C. IgE-binding is impaired in immunoblots, whereas in inhibition assays IgE binding to soluble Dau c 1.0401 is detected and it clearly provoked a response in mediator release assays., Conclusion: Dau c 1.0401 is a new isoallergen which contributes to the allergenicity of carrots. The absence of immunoreactivity in immobilized assays indicates that IgE binding is impaired when the protein is blotted on a solid phase. Altogether, the results point out that its allergenicity can be reduced upon carrot processing., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

About 50-70% of patients allergic to birch pollen suffer from sensitization after apple ingestion. Apple allergenicity was established in only few varieties. Studies were performed on apple fruits of 21 traditional and nine modern varieties organically, intensively, or integratively produced. The aim of the study was to assess whether the factors like cultivation method, maturity stage, genotype, or type of tissue place an impact on the allergenic potential of apples. To answer these questions, we used semiquantitative real-time PCR, ELISA, and immunoblotting. Apple allergen genes present divergent expression across apple cultivars. Expression of the Mal d 1.06A correlates with the Mal d 1 level and is affected by the cultivation method and maturity of the fruit. The content of the main allergen Mal d 1 varied widely across cultivars. Interestingly, in our study, the Gala variety presented a low Mal d 1 concentration regardless of the cultivation method. Based on the Mal d 1.06A expression, the Mal d 1 protein content, and the immunoreactivity assay, the Kandil Sinap, Kosztela, Rumianka from Alma-Ata, Kantówka Gdańska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

Almond (Prunus dulcis) is not only widely used as a human food as a result of its flavor, nutrients, and health benefits, but it is also one of the most likely tree nuts to trigger allergies. Almond allergens, however, have not been studied as extensively as those of peanuts and other selected tree nuts. This review provides an update of the molecular properties of almond allergens to clarify some confusion about the identities of almond allergens and our perspective on characterizing putative almond allergens. At present, the following almond allergens have been designated by the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-Committee: Pru du 3 (a non-specific lipid transfer protein 1, nsLTP1), Pru du 4 (a profilin), Pru du 5 (60S acidic ribosomal protein 2), Pru du 6 (an 11S legumin known as prunin) and Pru du 8 (an antimicrobial protein with cC3C repeats). Besides, almond vicilin and almond γ-conglutin have been identified as food allergens, although further characterization of these allergens is still of interest. In addition, almond 2S albumin was reported as a food allergen as a result of the misidentification of Pru du 8. Two more almond proteins have been called allergens based on their sequence homology with known food allergens and their 'membership' in relevant protein families that contain allergens in many species. These include the pathogenesis related-10 protein (referred to as Pru du 1) and the thaumatin-like protein (referred to as Pru du 2). Almonds thus have five known food allergens and five more likely ones that need to be investigated further. Published 2020. This article is a U.S. Government work and is in the public domain in the USA., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Scope: The major carrot allergen Dau c 1 belongs to the group of pathogenesis related class 10 (PR-10) proteins and is homologous to the birch pollen allergen Bet v 1. In contrast to most other PR-10 allergens, Dau c 1 can elicit Bet v 1 independent sensitization. Although Dau c 1 is considered heat labile, allergic reactions against cooked carrots are possible., Methods and Results: The pH and temperature stability as well as the allergenic potential before and after treatment of purified natural (n) Dau c 1 and different recombinant (r) isoallergens is investigated: rDau c 1.0104, rDau c 1.0105, rDau c 1.0201, rDau c 1.0301. All proteins except rDau c 1.0201 are able to refold at physiological pH. pH conditions around the pI (4.4-5.5) or the presence of the carrot matrix reduce the refolding capacity. Below the pI, most isoallergens are heat resistant and still able to cause mediator release, indicating allergenicity. Moreover, cooked carrot extract is still able to provoke mediator release due to remaining soluble Dau c 1., Conclusion: Patients allergic to carrots should avoid processed carrot containing foodstuff because heating or pH treatment do not completely abolish the allergenicity of Dau c 1., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Scope: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy., Methods and Results: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model., Conclusions: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity.

Allergy to pomegranate is often associated with severe symptoms. Two allergens have previously been described: 9k-LTP Pun g 1 and pommaclein Pun g 7. This study describes the isolation of a chitinase III, identified by direct protein sequencing and mass spectrometry. It is a 29-kDa protein showing 69% sequence identity with the latex hevamine and IgE binding in dot blotting, immunoblotting and FABER ® test. Chitinase-specific IgE were detected in 69 of 357 patients sensitized to one or more pomegranate allergenic preparations present on the FABER ® test. Using this test, 19.2% of the patients sensitized to kiwifruit chitinase IV were also sensitized to pomegranate chitinase III, rather than to latex chitinase I (7.2%) with which it shares the N-terminal hevein-like domain. In conclusion, a new allergen has been identified, contributing to improving food allergy diagnosis. This study reveals the important role of chitinases III and IV as allergy sensitizers and prompts further investigations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Birch pollen allergic patients show cross-reactivity to vegetables and fruits, including strawberries ( Fragaria × ananassa ). The objective of this study was to quantify the level of the Fra a 1 protein, a Bet v 1-homologous protein in strawberry fruits by a newly developed ELISA, and determine the effect of genotype, cultivation and food processing on the allergen amount. An indirect competitive ELISA using a specific polyclonal anti-Fra a 1.02 antibody was established and revealed high variability in Fra a 1 levels within 20 different genotypes ranging from 0.67 to 3.97 µg/g fresh weight. Mature fruits of red-, white- and yellow-fruited strawberry cultivars showed similar Fra a 1 concentrations. Compared to fresh strawberries, oven and solar-dried fruits contained slightly lower levels due to thermal treatment during processing. SDS-PAGE and Western blot analysis demonstrated degradation of recombinant Fra a 1.02 after prolonged (>10 min) thermal treatment at 99 °C. In conclusion, the genotype strongly determined the Fra a 1 quantity in strawberries and the color of the mature fruits does not relate to the amount of the PR10-protein. Cultivation conditions (organic and conventional farming) do not affect the Fra a 1 level, and seasonal effects were minor.

The strawberry fruit Fra a 1-proteins are homologues of the major birch pollen allergen Bet v 1 and have essential biological functions in pigment formation during fruit ripening. Patients affected by allergy against birch pollen tolerated fruits of a naturally occurring white-fruited F.×ananassa genotype, which showed reduced levels of Fra a 1 proteins along with enzymes of the anthocyanin pigment pathway. We evaluated the cross-reactive allergenic potential of a number of naturally occurring white- and red-fruited strawberry varieties to detect genotypes with low allergenic reactivity, whose fruit might be tolerated by patients with mild allergy. Protein extracts of 51 different strawberry varieties (Fragaria×ananassa, F. vesca, and F. nilgerensis) were screened by Western blot analysis with a polyclonal Fra a 1.02 antibody. Besides, activation of basophils of eight atopic patients allergic to birch pollen were studied using Bet v 1a and different concentrations of 15 selected strawberry protein extracts out of the 51 strawberry genotypes. Median percentages of activated basophils stimulated by extracts from white- and red-fruited genotypes ranged from 36 to 84% and 44 to 76%, respectively indicating that white-fruited strawberry are not per se hypoallergenic. Protein extracts from white-fruited F. vesca cv. Yellow Wonder showed the lowest cross-reactivity but high biological variability. The knowledge about the allergenic potential of different strawberry genotypes may help to improve food safety and can serve as starting point for the development of red-fruited hypoallergenic strawberry cultivars., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The consumption of pomegranate is increasing as it is considered a health-promoting food. Nevertheless, it can trigger allergic reactions, sometimes severe. The LTP Pun g 1 is the only pomegranate allergen so far reported. Based on preliminary clinical observations, the main aim of this study was the investigation of still unknown allergens contained in this fruit. Pommaclein, a homologue of peamaclein, the peach allergen Pru p 7, was isolated, identified by protein sequencing, and characterized as an IgE-binding protein by different test systems. RP-HPLC protein profiles revealed significant variations of LTP and pommaclein content in the red pulp of selected cultivars and accessions. Conversely, the mesocarp appeared free of proteins and much richer in antioxidants. In conclusion, a new allergen has been identified, and it could contribute to improving allergy diagnosis. The study highlights that pomegranate mesocarp could represent a rich and safe source of nutraceuticals also for allergic subjects.

Despite rapid growth of genetically modified (GM) crops, effective evaluations of genetic modification on allergenicity are still lacking. Gly m Bd 30K is cross-reactive with cow's milk protein casein, Gly m 4, and with birch pollen allergen Bet v 1. Here we compared the allergenicity between GM and non-GM soybeans with respect to the foci Gly m 4 and Gly m Bd 30K. Recombinant allergens of Gly m Bd 30K and Gly m 4 were generated and polyclonal antibodies raised to identify these two allergenic components in soybeans. GM soybean was first PCR-confirmed using 35S promoter. A total of 20 soybeans (half GM, half non-GM) obtained from a food market were used to assess their allergenicity based on IgE-binding and histamine release. The concentrations of Gly m Bd 30K and Gly m 4 in soybeans were then determined. Most soybean-allergic patients (9 of 10) showed IgE-positive reactions to the allergen of 30 kDa in molecular weight. That allergen turned out to be Glycine max Gly m Bd 30K based on LC-MS/MS analyses. Gly m Bd 30K is therefore the major allergen in the soybean. An increase in the transcription of both the Gly m 4 (stress-induced protein SAM22) and Gly m Bd 28K (soybean allergen precursor) was found after genetic modification. The protein concentrations of Gly m 4 and Gly m Bd 30K were not statistically significant different between non-GM and GM soybeans. There were also no statistical significances between them in the tests of IgE binding and histamine release. In conclusion, soybeans showed similar concentrations of Gly m Bd 30K and Gly m 4 regardless of genetic modification or absence thereof. The allergenicity of both Gly m Bd 30K and Gly m 4 was therefore not altered after genetic modification. Patients showing hypersensitivity to soybeans and who had pre-existing allergy to birch pollen and cow's milk casein might not further increase their allergic reactions following exposures to the GM soybeans.

The strawberry fruit proteins Fra a 1.01E-1.08 are homologues of the major birch pollen allergen Bet v 1. Three of the proteins are known to have essential biological functions in pigment formation during fruit ripening and seem to be responsible for allergic reactions to strawberry fruit. We evaluated the cross-reactive allergenic potential of these putative strawberry allergens in patients allergic to birch pollen. Activation of basophils of eight atopic patients was studied using different concentrations of Fra a 1 isoforms. Bet v 1a was used as control and as atopic patient selection criterion. Although Fra a 1.01E-1.08 have amino acid sequence identities of 74.5-97.5% with Fra a 1.02, the basophil activation mediated by the eight Fra a 1 proteins differed substantially. Fra a 1.03 and Fra a 1.02 showed the highest activation of basophils, 73 and 66% of total basophils, respectively. On the basis of the high relative expression of the gene Fra a 1.02 in ripe strawberry fruits of allergenic varieties, Fra a 1.02 was identified as the main strawberry allergen of the Bet v 1 superfamily. Knowledge of the allergenic potential of Fra a 1.02/1.03 will help to improve food safety and can serve as a valuable marker for the development of red-fruited hypoallergenic strawberry cultivars.

Background: Non-specific lipid transfer proteins (nsLTP) are considered to provoke allergic symptoms to plane tree pollen, which are frequently associated with peach allergy., Objective: The objective was to clone the cDNA of plane pollen nsLTP Pla a 3, to characterize IgE-binding and allergenic potency of recombinant Pla a 3 in comparison to its natural counterpart and peach nsLTP Pru p 3., Methods: Natural Pla a 3 was purified from plane pollen and analysed by mass spectrometry (MS). Recombinant Pla a 3 was characterized by SDS-PAGE and CD spectroscopy. Specific IgE to extract, components of plane pollen and Pru p 3 was measured by ImmunoCAP in sera of patients allergic to either plane pollen (n = 10), peach (n = 15) or both (n = 15). Biological potency of the proteins was investigated by in vitro mediator release assays and IgE cross-reactivity by competitive ELISA., Results: Two Pla a 3 isoforms were identified. Recombinant Pla a 3 showed high purity, structural integrity, IgE-binding capacity comparable to nPla a 3 and biological potency. Sensitization to plane pollen extract was confirmed in 24/25 plane pollen allergics. The frequency of sensitization to Pla a 3 was 53% among patients allergic to both plane pollen and peach and 10% among plane pollen allergics tolerating peach where most patients were sensitized to Pla a 1. Pla a 3 and Pru p 3 showed strong bi-directional IgE cross-reactivity in patients allergic to peach and plane pollen, but not in peach allergics tolerating plane pollen. Levels of IgE-binding were generally higher to Pru p 3 than to Pla a 3., Conclusion: Sensitization to Pla a 3 is relevant in a subgroup of plane pollen allergics with concomitant peach allergy. IgE testing with Pla a 3 may serve as a marker to identify plane pollen allergic patients at risk of LTP-mediated food reactions and thereby improve in vitro diagnostic procedures., (© 2016 John Wiley & Sons Ltd.)

Background: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals., Methods: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS)., Results: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2., Conclusion: These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Scope: The aim was to investigate the potential contribution of a major birch pollen Bet v 1-homologous allergen to birch pollen-associated tomato fruit allergy., Methods and Results: Two isoforms of a Bet v 1-homologous protein (designated Sola l 4) from tomato fruit were identified by cDNA-cloning and produced as recombinant proteins. Allergen-specific IgE levels to tomato, birch pollen, Bet v 1, and Sola l 4 were determined in birch pollen allergic patients with allergy or tolerance to tomato. Sola l 4 was recognized in 76% of birch/tomato allergic patients, while tomato- and Bet v 1-specific IgE was detectable in 64% and 81% of sera. Almost all patients sensitized to Bet v 1 reacted with Sola l 4. Both Sola l 4 isoforms displayed allergenic potency and IgE-cross-reactivity with Bet v 1 as investigated by competitive ELISA and in vitro mediator release assay. Nevertheless, the reactivity pattern of patients' sera was diverse., Conclusion: Sola l 4, a novel pathogenesis related-10 protein, qualifies as major allergen in tomato fruits. Data suggest Sola l 4 as class II allergen. IgE-testing using Sola l 4 showed low clinical specificity, but high sensitivity in tomato allergic patients and will further improve component-resolved allergy diagnosis., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.

Background: Allergy to sapodilla (Manilkara zapota) fruit ingestion is rare. An independent study from our group has identified a basic thaumatin-like protein (TLP 2) as the major allergen. The present study was aimed at identifying and characterizing additional allergens from sapodilla., Methods: Allergic subjects were identified by case history, skin prick test (SPT) and allergen-specific IgE. Sapodilla extract was fractionated using SP-Sepharose into 3 components (SP1, SP2 and SP3) which were analyzed by native/SDS-PAGE, IgE-immunoblot, isoelectric focusing (IEF) and N-terminal sequencing. The conserved regions of plant TLPs and the N-terminal sequence were used to design primers for PCR., Results: SPT and ELISA confirmed a subject with oral allergy syndrome (OAS) to sapodilla and custard apple. Two proteins (26.9 and 24.5kDa; reducing conditions) were detected as allergens, of which the latter in SP2 has already been identified as basic TLP (TLP 2). The 26.9kDa protein present in SP1 was identified as an acidic TLP based on native PAGE, IEF and N-terminal sequencing. Presence of a basic β-1,3-glucanase in SP3 was inferred by zymography. Sequence analysis of the genomic clone of the acidic TLP gene revealed that it is intronless and non-glycosylated. Evolutionary relatedness to olive, grape and kiwi fruit allergenic TLPs were inferred by phylogenetic analysis., Conclusions: An acidic TLP (TLP 1) was identified as a new allergen in sapodilla. TLP 1 is a single polypeptide (207 residues) belonging to the thaumatin family of the GH64-TLP-SF superfamily. Clinically, sapodilla should be considered in the list of fruits causing OAS.

Rice, the staple food of south and east Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16, 26, 33, and 56 kDa have been identified as allergens. Recently, it was documented that the 56 kDa rice allergen was responsible for rice-induced anaphylaxis. The 14-16 kDa allergens have been identified as α-amylase inhibitors; the 26 kDa protein has been identified as α-globulin; and the 33 kDa protein has been identified as glyoxalase I. However, the identity of the 56 kDa rice allergen has not yet been determined. In this study, we demonstrate that serum from patients allergic to maize shows IgE binding to a 56 kDa protein that was present in both maize and rice but not in the oil seeds soybean and peanut. The 56 kDa IgE-binding protein was abundant in the rice endosperm. We have purified this protein from rice endosperm and demonstrated its reactivity to IgE antibodies from the serum of maize-allergic patients. The purified protein was subjected to matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry analysis, resulting in identification of this rice allergen as granule-bound starch synthase, a product of the Waxy gene. Immunoblot analysis using protein extracts from a waxy mutant of rice revealed the absence of the 56 kDa IgE-binding protein. Our results demonstrate that the 56 kDa rice allergen is granule-bound starch synthase and raise the possibility of using waxy mutants of rice as a potential source of the hypoallergenic diet for patients sensitized to the 56 kDa rice allergen.

Scope: Manioc (Manihot esculenta) is a tuber mainly consumed in the Southern Hemisphere and used worldwide by food and chemistry industry. We aimed to recombinantly produce and characterize the first manioc allergen and evaluate its IgE reactivity in sera of Brazilian and Italian patients., Methods and Results: The molecule, termed Man e5, was expressed in E. coli, characterized by amino acid analysis, mass spectrometry, circular dichroism, HPLC, and dynamic light scattering. A tertiary structural model of the protein was produced using bioinformatics and susceptibility to pepsin digestion was analyzed in vitro. Based on its high content of charged residues, heat stability, flexibility and lack of secondary structure elements, the allergen was determined a member of the intrinsically disordered protein family. Brazilian patients were selected based on manioc allergy and Italians based on latex allergy and sensitization to Hev b 5.71% of Brazilians and 40% of Italians were in vitro IgE positive to Man e5. Cross-inhibition assays suggest a possible involvement of this allergen in the latex-fruit syndrome., Conclusion: Man e5, the first purified allergen from manioc demonstrates IgE cross-reactivity with Hev b 5. Data suggest Hev b 5 might act as primary sensitizer and could therefore lead to allergic manifestations upon manioc consumption without prior exposition., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: After consumption of fruits, nuts, and vegetables, several patients with pollen allergy experience gastrointestinal (GI) tract symptoms that are possibly caused by pollen-associated food allergy. The aim of this study was to evaluate the colonoscopic allergen provocation (COLAP) test using the recombinant birch pollen allergen Bet v 1 (rBet v 1) for in vivo diagnosis of pollen-associated food allergy manifesting in the GI tract., Methods: Thirty-four patients with a history of adverse reactions to food, GI tract symptoms, and birch pollen pollinosis and five healthy controls underwent COLAP test. Twenty minutes after endoscopic challenge of the cecal mucosa with rBet v 1, the mucosal wheal and flare reaction was registered semiquantitatively, and tissue biopsy specimens were examined for eosinophil mucosal activation., Results: The mucosal reaction to rBet v 1 was correlated with the presence of pollinosis (P = 0.004), history of adverse reaction to Bet v 1-associated food allergens (P = 0.001), and tissue eosinophils' activation (P < 0.001). A wheal and flare reaction in the COLAP test was observed in 13 of 16 patients (81%) with a history of GI tract symptoms associated with the ingestion of Bet v 1-related foods and in four of 18 (22%) patients with a negative history (P < 0.001). The control group did not develop visible mucosal reactions to rBet v 1. Systemic anaphylactic reactions did not occur., Conclusions: The mucosal administration of rBet v 1 by COLAP test provides a new diagnostic tool that might support the diagnosis of Bet v 1-associated food allergy manifesting in the GI tract., (© 2012 John Wiley & Sons A/S.)

Selected members of plant pathogenesis-related and seed storage proteins represent specific groups of proteins with potential characteristics of allergens. Efforts to understand the mechanism by which pathogenesis-related proteins mediate a broad cross-reactivity in pollen-plant food allergens are still limited. In this study, computational biology approach was used to reveal specific structural implications and conservation of different epitopes from members of Bet v 1 and nsLTP protein families mediating cross-reactivity between pollen and food (fruits, vegetables, legumes, and nut/seeds) allergens. A commonly shared epitope conservation was found among all pollen and food Bet v 1 and nsLTP protein families, respectively. However, other allergenic epitopes were also specifically detected in each family. The implication of these conserved epitopes in a broad cross-reactivity for allergy clinical trials is here discussed.

Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.

Scope: Four Bet v 1 homologous food allergens from celeriac (rApi g 1), apple (rMal d 1), peach (rPru p 1) and hazelnut (rCor a 1), were used to probe the structural responsiveness of the Bet v 1 scaffold to gastric digestion conditions and its impact on allergenicity., Methods and Results: Low pH induced conformational changes of all homologues, which was reduced at physiological ionic strength for all except rPru p 1 as observed by circular dichroism (CD)-spectroscopy. The homologues were rapidly digested by pepsin, losing their IgE binding activity, although the kinetics and patterns of digestion varied subtly between homologues, rApi g 1 being the most stable. We have demonstrated for the first time that gastric phosphatidyl-choline (PC) induced conformational changes in all homologues but only rMal d 1 penetrated the PC vesicles as detected by fluorescence polarization, slowing its digestion and retaining more of its allergenic activity. PC enhanced basophil activation of all digested allergens except rApi g 1., Conclusion: The Bet v 1 scaffold is generally susceptible to low pH and pepsinolysis and interacts with PC vesicles, properties which can explain effects of the gastric environment on their allergenicity. These data show the importance of including surfactants in model digestion systems., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Arbuscular mycorrhizal (AM) fungi influence the expression of defence-related genes in roots and can cause systemic resistance in plants probably due to the induced expression of specific defence proteins. Among the different groups of defence proteins, plant food allergens were identified. We hypothesized that tomato-allergic patients differently react to tomatoes derived from plants inoculated or not by mycorrhizal fungi. To test this, two tomato genotypes, wild-type 76R and a nearly isogenic mycorrhizal mutant RMC, were inoculated with the AM fungus Glomus mosseae or not under conditions similar to horticultural practice. Under such conditions, the AM fungus showed only a very low colonisation rate, but still was able to increase shoot growth of the wild-type 76R. Nearly no colonisation was observed in the mutant RMC, and shoot development was also not affected. Root fresh weights were diminished in AM-inoculated plants of both genotypes compared to the corresponding controls. No mycorrhizal effects were observed on the biomass and the concentration of phosphate and nitrogen in fruits. Real-time quantitative polymerase chain reaction analysis revealed that six among eight genes encoding for putative allergens showed a significant induced RNA accumulation in fruits of AM-colonised plants. However, human skin reactivity tests using mixed samples of tomato fruits from the AM-inoculated and control plants showed no differences. Our data indicate that AM colonisation under conditions close to horticultural practice can induce the expression of allergen-encoding genes in fruits, but this does not lead necessarily to a higher allergenic potential.

Pathogenesis-related protein-10 (PR10) is a ubiquitous small plant protein induced by microbial pathogens and abiotic stress that adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. In this plant, two highly similar genes encoding PR10 isoforms have been isolated and designated as allergen Dau c 1.01 and Dau c 1.02. The aim of the study was to generate PR10-reduced hypoallergenic carrots by silencing either one of these genes in transgenic carrots by means of RNA interference (RNAi). The efficiency of gene silencing by stably expressed hairpin RNA (hnRNA) was documented by means of quantitative RT-PCR (qPCR) and immunoblotting. Quantification of the residual protein revealed that PR10 accumulation was strongly decreased compared with untransformed controls. Treatment of carrot plants with the PR protein-inducing chemical salicylic acid resulted in an increase of PR10 isoforms only in wild-type but not in Dau c 1-silenced mutants. The decrease of the allergenic potential in Dau c 1-silenced plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests (SPT). However, simultaneous silencing of multiple allergens will be required to design hypoallergenic carrots for the market. Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This constitutes a reasonable approach to allergen avoidance.

Some GM crops including MON863 corn and stack varieties contain Cry3Bb1 protein. Cry3Bb1 is very important from the standpoint of assessing the safety of GM crops. In this study Cry3Bb1 was assessed from the standpoint of possible binding to IgE from allergy patients. First, an ELISA that was improved in our laboratory was used to test serum samples from 13 corn allergy patients in the United States with recombinant Cry3Bb1 expressed in Escherichia coli, and serum samples from 55 patients in Japan with various food allergies were also assayed. Two samples from the Japanese allergy patients were suspected of being positive, but Western blotting analysis with purified Cry3Bb1 indicated that the binding between IgE and Cry3Bb1 was nonspecific. Ultimately, no specific binding between IgE and recombinant Cry3Bb1 was detected. Next, all proteins extracted from MON863 corn and non-GM corn were probed with IgE antibodies in serum samples from the corn allergy patients by Western blotting, but the staining patterns of MON863 and non-GM corn were similar, meaning that unintended allergic reactions to MON863 are unlikely to occur. Our study provides additional information that confirms the predicted lack of IgE binding to Cry3Bb1 in people with existing food allergies., (Copyright 2009 Elsevier Inc. All rights reserved.)

The development of techniques devised for the genetic manipulation of foods poses new risks for children with food allergy (FA). The introduction of foreign allergenic proteins from different foods into previously tolerated foods may trigger allergic reactions, often complicating with anaphylactic shock in a subset of allergic babies. Children with FA, even if subjected to preventative diets, always challenge the risk of developing allergic manifestations after unintentional intake of a non tolerated food in restaurant settings, with relatives or schoolmates, etc, where product labelling is necessarily lacking. The introduction of potentially allergenic proteins into foods generally considered safe for allergic children can be done deliberately, by either substantially altering the food ingredients, or by genetic manipulation which change the composition or transfer allergens, or unintentionally by qualitycontrol failures, due to contaminations in the production process, or to genetic mismanipulation. There is a controversy between multinationals often favored by governments and consumer association resistance, thus an equidistant analysis poses some unprecedented impediments. The importance of FA and the potential of transgenic plants to bring food allergens into the food supply should not be disregarded. The expression in soybeans of a Brazil nut protein resulted in a food allergen ex-pressed in widely used infant formulas, so paving the way to an often reported multinational debacle. Genetic engineering poses innovative ethical and social concerns, as well as serious challenges to the environment, human health, animal welfare, and the future of agriculture. In this paper will be emphasized practical concepts more crucial for pediatricians.

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.

Background: The 16-kDa protein of buckwheat (BW) has been implicated as a major allergen in BW allergy., Objective: To characterize the 16-kDa allergen and evaluate its clinical significance as an indicator of BW allergy., Methods: Complementary DNA from the 16-kDa allergen was cloned and expressed in Escherichia coli. Allergenicity was confirmed with IgE immunoblotting or with an enzyme-linked immunosorbent assay. The clinical utility of the recombinant protein (r16 kDa) for diagnosis of BW reactivity was evaluated in 18 BW-allergic and in 20 asymptomatic BW-sensitized subjects., Results: The 16-kDa allergen, composed of 127 amino acids, has 50% homology to the reported 8-kDa BW allergen, which belongs to the 2 S storage albumin. The r16-kDa protein can inhibit specific IgE (sIgE) antibody binding to the native BW 16-kDa allergen but minimally inhibited sIgE binding to crude BW extract. Approximately 77.8% of patients with the BW allergy produced sIgE antibodies to the r16-kDa protein, compared with a complete lack of reactivity in the 20 asymptomatic BW-sensitized subjects. The areas of the receiver operating characteristic curves for the skin prick test (mean, 0.93; 95% confidence interval, 0.85 to approximately 1.01; P < .001) and the rl6-kDa enzyme-linked immunosorbent assay (mean, 0.93; 95% confidence interval, 0.84 to approximately 1.01; P < .001) were higher than the area of the BW IgE measurement curve determined by ImmunoCAP (a system for assaying serum IgE) (mean, 0.80; 95% confidence interval, 0.66 to approximately 0.94; P = .002)., Conclusions: The 16-kDa allergen belongs to the 2 S storage albumin. Measurement of rl6-kDa sIgE was more discriminating than measurement of ImmunoCAP sIgE in whole BW extracts for the diagnosis of clinical reactivity to BW.

Background: Sesame allergy is increasingly being reported, and multi-sensitization to peanut and tree nuts has been described. The clinical relevance and cross-reactivity of many sesame proteins, such as Ses i 6, are unknown., Objective: The aims of this study were to perform a preliminary examination of the cross-reactivity of Ses i 6 in vitro, examine the ability of Ses i 6 to activate basophils in a modified basophil activation test (mBAT), and assess whether such an assay may help to distinguish between potentially relevant and irrelevant IgE reactivity towards 11S globulin proteins., Methods: Inhibition immunoblotting and chicken anti-rJug r 4 antibodies were used to determine the cross-reactivity of rSes i 6. Basophils from atopic donors were stripped of resident IgE before passive sensitization with food-allergic sera and challenged with protein extracts or recombinant protein. Basophil activation was measured using two activation markers, CD203c and CD63, via flow cytometry., Results: IgE immunoblotting showed cross-reactivity between rJug r 4 and rSes i 6 using sera from two human donors and chicken IgY. Additionally, rSes i 6 activated basophils passively sensitized with sesame-allergic sera. Cross-reactive serum from a sesame-allergic but walnut-tolerant donor was not able to activate basophils when challenged by walnut extract despite IgE reactivity to walnut determined by immunoblotting., Conclusions: The sesame 11S globulin shows partial immunological cross-reactivity with walnut, and although it is classified as a minor allergen, activated basophils sensitized with serum from seven out of eleven sesame-allergic donors. Additionally, the mBAT may help distinguish between clinically relevant and irrelevant in vitro IgE cross-reactivity of seed storage proteins in nuts and seeds and thus warrants use in further studies.

Background: IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis., Objective: Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3., Methods: Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay., Results: Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays., Conclusion: Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.

Background: In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya versus its non-transgenic control., Methods: We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples., Results: We identified 2 new potential soybean allergens--one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study., Conclusion: Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment., (Copyright (c) 2007 S. Karger AG, Basel.)

Background: The identity of allergenic almond proteins is incomplete., Objective: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen., Methods: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin., Results: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins., Conclusion: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients., Clinical Implications: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

Background: Today, for patients with food allergy, the only possibility to prevent allergic reactions is avoidance of the allergenic food. Genetic engineering of hypoallergenic plants by means of RNA interference (RNAi) could be an approach to improve the quality of life of subjects with food allergy., Objectives: We sought to achieve stable inhibition of expression of the allergenic nonspecific lipid transfer protein Lyc e 3 in tomato and to analyze the reduction of allergenicity in vitro by using histamine release assays and in vivo by using skin prick tests with transgenic tomato fruits., Methods: Gene silencing was performed by means of RNAi and monitored by using Western blotting with nonspecific lipid transfer protein-specific antibodies and sera from patients with tomato allergy. Dose-dependent basophil histamine release assays, prick-to-prick skin testing, and determination of endogenous histamine content were performed with fruits harvested from plants of the first and second generation to assess the allergenic potency compared with that of wild-type fruits., Results: We demonstrated that silencing of Lyc e 3 by means of RNAi contributes to reduced skin reactivity and is passed on to the next generation of fruits. A significant reduction of allergenic potency was determined in vitro and confirmed by using skin prick tests., Conclusion: Taken together, these results indicate that RNAi technology is an effective tool to generate foods with reduced allergenicity., Clinical Implications: Allergen-reduced plant foods might allow reduction of dietary restrictions for patients allergic to panallergen families.

Background: Chestnut allergy has been almost exclusively considered in the context of the latex-fruit syndrome. Chestnut allergens not linked to latex hypersensitivity have not been studied., Objective: We sought to explore whether differences in sensitization patterns between chestnut allergy with or without associated latex-fruit syndrome can be detected., Methods: Twelve patients sensitized to chestnut but not to latex and 3 control patients with latex-chestnut allergy were analyzed. A major chestnut allergen was purified and characterized. IgE immunoblotting, specific IgE determination, and skin prick tests with 5 isolated allergens involved in food allergy or latex-fruit syndrome were also performed., Results: A major 9-kd allergen was detected in chestnut extract, isolated, and identified as lipid transfer protein (LTP) Cas s 8. Specific IgE to this allergen was found in 91% (by means of IgE immunoblotting) and 58% (by means of ELISA) of sera from patients with chestnut but not latex allergy. Moreover, 66% of these patients had positive skin prick test responses to Cas s 8. Additionally, allergenic LTPs from peach fruit and Artemisia vulgaris pollen were also reactive. In contrast, avocado class I chitinase and latex hevein, allergens associated with the latex-fruit syndrome, showed no reaction. The opposite situation was exhibited by patients with latex-chestnut allergy., Conclusions: Patients with chestnut allergy with or without associated latex hypersensitivity present different patterns of major allergens (LTPs and class I chitinases, respectively)., Clinical Implications: LTPs and class I chitinases can be used as diagnostic tools in patients with chestnut allergy to predict whether an associated latex sensitization and a risk of potential cross-reactivity with other plant foods and pollens exist.

Background: In contrast to other Rosaceae fruit, only few cases of patients with adverse reactions to strawberry are listed in literature. OBJECTIVE To identify allergenic proteins in strawberry and to express and characterize recombinant strawberry lipid transfer protein (LTP; rFra a 3)., Methods: Established apple-allergic patients were recruited on the basis of a reported allergic reaction to strawberry (n=28, confirmed by double-blind placebo-controlled food challenge in four patients) or on the basis of IgE reactivity to LTP (n=34). Sensitization to purified natural and recombinant allergens was assessed by RAST, immunoblot (inhibition) and basophil histamine release (BHR). A strawberry cDNA library was screened for genes homologous to known fruit allergens. Fra a 3 was cloned and expressed in the yeast Pichia pastoris and compared with peach and apple LTP by RAST, immunoblot-inhibition and BHR tests., Results: Genes homologous to Bet v 1, Bet v 6, profilin and LTP were identified in a strawberry cDNA library. In BHR the rFra a 3 induced histamine release at a 100-fold higher concentration than peach LTP. RAST inhibition showed high cross-reactivity to peach and apple LTP, although IgE reactivity was lower by a factor 5. On strawberry immunoblot, patients' IgE showed reactivity to a Bet v 1 homologue, profilin, LTP and high-molecular weight bands., Conclusion: In addition to a Bet v 1 homologue, strawberry also contains IgE-binding profilin and LTP. The rFra a 3 has less allergenic potency than peach and apple LTP, and therefore is an interesting tool for future immunotherapy. Fra a 3 does not seem to be clinically relevant.

The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were beta-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that the rin gene has the potential to regulate allergen accumulation in tomato fruit.