Molecular and Immunological Identification of Low Allergenic Fruits among Old and New Apple Varieties.

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 Gdanska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

Birch pollen immunotherapy inhibits anaphylaxis to the cross-reactive apple allergen Mal d 1 in mice.

BACKGROUND: Cross-reactive apple allergy is a common co-morbidity of birch pollen allergy, caused by the presence of a Bet v 1 homologue allergen in apple, Mal d 1. Treatment of tree pollen hay fever by immunotherapy is well established, but its effect on the accompanying apple allergy is debated. OBJECTIVE: To establish a mouse model of birch pollen induced cross-reactivity to Mal d 1 and investigate the effect of birch pollen immunotherapy on the cross-reactivity to Mal d 1. METHODS: Respiratory allergy was induced in Balb/c mice by intraperitoneal exposure to alum-adsorbed birch pollen extract (BPE) in combination with short or prolonged intranasal exposure to BPE. To evaluate the response to Mal d 1, mice were exposed intraperitoneally to Mal d 1. Immunoglobulin responses and cytokine production by splenocytes were measured by ELISA. Allergic symptoms were evaluated by measuring airway hyper-reactivity and hypothermia as a surrogate marker for anaphylaxis. Immunotherapy was performed subcutaneously with alum-adsorbed BPE. RESULTS: Mice exposed to BPE develop cross-reactive IgE to Mal d 1. Early after exposure to BPE, this response is still weak and does not yet translate into anaphylaxis. Interestingly, later re-challenge with BPE increased cross-reactivity to a level where Mal d 1 exposure induced anaphylaxis. Cross-sensitization can also be induced by systemic Mal d 1 exposure. Birch pollen immunotherapy significantly reduced the anaphylactic response of mice to Mal d 1. CONCLUSION & CLINICAL RELEVANCE: A mouse model mimicking birch pollen induced cross-reactivity to Mal d 1 was successfully established. In this model, birch pollen immunotherapy significantly ameliorated the anaphylaxis induced by Mal d 1. Our experimental data suggest that boosting of Mal d 1 recognizing immunoglobulins by BP SCIT is important for the amelioration of apple allergy in human.

First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.

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.

Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site-directed mutagenesis.

BACKGROUND: Birch pollen allergies are frequently associated with adverse reactions to various fruits, nuts, or vegetables, described as pollen-food syndrome (PFS) and caused by cross-reactive IgE antibodies primarily directed against Bet v 1. Specific immunotherapy (SIT) represents an effective treatment for inhalant allergies; however, successful birch pollen SIT does not correlate well with the amelioration of concomitant food allergies. METHODS: As vaccine candidates, apple Mal d 1 as well as hazelnut Cor a 1 derivatives were designed by in silico backbone analyses of the respective allergens. The proteins were produced by site-directed mutagenesis as fold variants of their parental allergens. Because Mal d 1 and Cor a 1 form cysteine-mediated aggregates, nonaggregative cysteine to serine mutants were also generated. The proteins were characterized physicochemically, immunologically, and in in vivo models with or without adjuvant. RESULTS: The structurally modified proteins showed significantly decreased IgE binding capacity. Notably, both in vivo models revealed reduced immunogenicity of the hypoallergenic fold variants. When formulated with alum, the monomeric cysteine mutants induced a similar immune response as the aggregated parental allergens, which is in contrast with data published on Bet v 1. CONCLUSION: These findings lead to the suggestion that the Bet v 1 structure has unique intrinsic properties, which could account for its high allergenicity. Obviously, these characteristics are not entirely shared with its food homologues from apple and hazelnut. Thus, it is important to tackle pollen-related food allergies from different angles for the generation of effective vaccine candidates to treat birch PFS.

Effects of long-term storage on Mal d 1 content of four apple cultivars with initial low Mal d 1 content.

BACKGROUND: Up to 6.6% of adults and 11.5% of children aged between 0 and 6 years are reporting a perceived fruit allergy. In the last group 8.5% perceived reactions against apple. To keep allergic reactions as low as possible and to make apple consumption more tolerable for patients allergic to apple, the Mal d 1 content of four apple cultivars ('Boskoop', 'Elise', 'Pinova', 'Santana' and 'Topaz') were analyzed, to identify apple cultivars with only a low Mal d 1 content. Further, the development of Mal d 1 content of low-allergenic cultivars should be investigated during long-term storage. RESULTS: The lowest Mal d 1 content was measured for fruits of the cultivar 'Santana' at harvest, followed by 'Pinova' and 'Elise' after storage without 1-MCP. Nearly all fruits treated with 1-MCP had a significantly lower Mal d 1 content than fruits without 1-MCP, except for 'Boskoop' fruits after 44 weeks of storage. Apple cultivars with a low Mal d 1 content are not a cross of 'Golden Delicious' with another cultivar, with the exception of 'Pinova' which is a cultivar from the so-called 'Pi-series', which have a moderate resistance to scab and mildew. First experiments on tomatoes and peanuts have shown a reduction of allergic proteins in older and child generations after transfer of RNA. This reduction is probably based on an alteration of gene expression regulating the synthesis of allergic proteins, leading to changes in protein composition in the fruit. CONCLUSION: Individuals allergic to apple should choose the described cultivars with relative low Mal d 1 content. If breeders want to breed cultivars with low allergy, they should avoid crossings with 'Golden Delicious'.

Profiles of apple allergen components and its diagnostic value in Northern China.

Background: Limited is known on the profiles of apple allergy in China. Objective: To explore the clinical significance of apple allergen components in northern China. Methods: This study recruited 40 participants and categorized into apple tolerance (n = 19) and allergy (n = 21) group. The latter was categorized into oral allergy symptoms (OAS, n = 14) and generalized symptoms (GS, n = 7). All participants underwent ImmunoCAP screening to assess sIgE levels of birch, apple, and their components. Results: The sensitization rates were 90% for Bet v 1, 85% for Mal d 1, 35% for Bet v 2, and 20% for Mal d 3. The overall positive rate for apple allergens was 97.5%, with half demonstrating mono-sensitization to Mal d 1. Birch, Bet v 1 and Mal d 1 sIgE levels had consistent areas under the curve (AUC 0.747, p = 0.037; AUC 0.799, p = 0.012; AUC 0.902, p < 0.001 respectively) in diagnosing apple allergy. The optimal cut-off values were determined to be 22.85 kUA/L (63.6% sensitivity, 85.7% specificity), 6.84 kUA/L (81.8% sensitivity, 71.4% specificity) and 1.61 kUA/L (93.8% sensitivity, 75.0% specificity), respectively. No allergens or components demonstrated diagnostic value in distinguishing between OAS and GS. Mal d 3 sensitization was correlated with mugwort allergy and higher risk of peach, nuts or legumes generalized allergy. Conclusion: Mal d 1 was major allergen and the best for diagnosing apple allergy. Mal d 3 does not necessarily indicate severe allergic reaction to apples in northern China but may indicate mugwort sensitization and an increased risk of peach, nuts or legumes allergy.

Multianalytical Approach to Understand Polyphenol-Mal d 1 Interactions to Predict Their Impact on the Allergenic Potential of Apples.

Interactions between phenolic compounds and the allergen Mal d 1 are discussed to be the reason for better tolerance of apple cultivars, which are rich in polyphenols. Because Mal d 1 is susceptible to proteolytic digestion and allergenic symptoms are usually restricted to the mouth and throat area, the release of native Mal d 1 during the oral phase is of particular interest. Therefore, we studied the release of Mal d 1 under different in vitro oral digestion conditions and revealed that only 6-15% of the total Mal d 1 present in apples is released. To investigate proposed polyphenol-Mal d 1 interactions, various analytical methods, e.g., isothermal titration calorimetry, 1H-15N-HSQC NMR, and untargeted mass spectrometry, were applied. For monomeric polyphenols, only limited noncovalent interactions were observed, whereas oligomeric polyphenols and browning products caused aggregation. While covalent modifications were not detectable in apple samples, a Michael addition of epicatechin at cysteine 107 in r-Mal d 1.01 was observed.

Where to prick the apple for skin testing?

Mal d 1 is not equally distributed over the apple. We aimed to examine the influence of the location of pricking in the apple on prick-to-prick skin prick test (PTP) results. PTPs were performed in autumn 2007 and spring 2008, before the birch pollen season, in 32 Dutch adults with symptoms of oral allergy to fresh apple, using apples harvested in autumn 2007. PTPs with fresh intact and unpeeled Pink Lady, Golden Delicious, Elise, Santana and Modi apples were performed using material obtained from approximately 2 cm near the stalk (top), and the middle region. All PTP responses were greater when performed with apple material near the stalk than from the middle region. In 2007, these differences were statistically significant for Pink Lady, Golden Delicious and Elise, and in 2008, for Pink Lady and Modi. When performing PTPs, the apple should be pricked near the stalk rather than in the middle.

Analyzing Bioaccessibility of Polyphenols in Six Commercial and Six Traditional Apples (Malus domestica Borkh.) during In Vitro and Ex Vivo Oral Digestion.

SCOPE: Apples are an important polyphenol (PP) source. To compare the health benefits of traditional and commercial varieties, the phenolic contents and profiles as well as their release from the matrix (bioaccessibility) during oral digestion are determined. Furthermore, based on these data the proposed beneficial effect of PP on the variety specific allergenicity is discussed. METHODS AND RESULTS: Phenolics are quantified by HPLC-DAD. Total phenolic contents (TPC) are in the range of 111-645 and 343-1950 mg 100 g-1 dry weight for flesh and peel, respectively. Matrix release during oral digestion is investigated ex vivo, with centrifuged and non-centrifuged human saliva and in vitro with simulated saliva fluid (SSF). The overall bioaccessibility is similar in all digestion media, ranging between 40-80% and 39-65% of the TPC in flesh and peel, respectively. Analyzing the correlation among Mal-d 1-allergen-content, unoxidized PP, and the allergenic potential for the samples reveals a negligible effect of phenolics. CONCLUSION: Due to higher phenolic contents in combination with a similar release, increased PP concentrations in the oral phase and an improved uptake of PP from traditional varieties are assumed. However, the proposed beneficial effect of phenolics on allergenicity cannot be confirmed.

Impact of Cultivation and Storage Conditions on Total Mal d 1 Content and Isoallergen Profile in Apples.

The allergen Mal d 1 is often responsible for adverse allergic reactions to fresh apples in northern and central Europe. The Mal d 1 content and isoallergen profile are proposed to have an impact on the allergenic potential of the fruit. Therefore, we investigated the impact of the cropping system on the Mal d 1 content and the isoallergen profile of apples by mass spectrometry for the varieties 'Jonagored' and 'Topaz'. To monitor the impact of storage time and conditions, apples of the varieties 'Santana' and 'Jonagold' were stored for up to 12 weeks under regular air (RA), under RA in combination with 1-methylcyclopropene (1-MCP) treatment, and under a controlled atmosphere (CA). The impact of the cropping system (integrated production vs organic production) was negligible. However, a significant increase in the Mal d 1 content during storage was observed, being higher when stored under CA conditions than under RA conditions. An additional treatment with 1-MCP prior to RA storage drastically reduced the level of Mal d 1 expression in the flesh of the apples by ∼50%. Furthermore, the content of isoallergens 1.03 and 1.06 increased disproportionately under CA conditions, while under RA conditions, only isoallergen 1.06 was affected. With the 1-MCP treatment, no changes in the isoallergen profile were obvious.

Characterization and Quantification of Mal d 1 Isoallergen Profiles and Contents in Traditional and Commercial Apple Varieties by Mass Spectrometry.

The apple allergy in Northern Europe is a cross-reaction to the birch pollen allergy. No correlation between the allergenicity of an apple variety and the content of the major apple allergen Mal d 1, a homologue to the Bet v 1 allergen in birch, could be found using ELISA, so far. Therefore, an impact of polyphenols and/or differences in the isoallergen profile are discussed. To allow a more detailed analysis of the Mal d 1 content and the isoallergen profile, a mass spectrometric method was applied to investigate differences in the flesh and peel of 10 traditional varieties and 10 commercial breeds. The data revealed often, but not always, lower Mal d 1 contents in traditional varieties grown in orchard meadows, which was more obvious in the flesh. Differences among the peels were less pronounced. A closer look at the individual isoallergens 1.01, 1.02, 1.03, and 1.06 reveals an increased impact of the minor isoallergens 1.03 and 1.06 on the allergenic potential, since commercial breeds like Braeburn, Santana, and Holstein Cox, which are considered to have reduced allergenic potentials, were characterized by low levels of these isoallergens.

Profiles of Birch Allergen Component Sensitization and Its Association with Pollen Food Allergy Syndrome in Northern China.

Purpose: To investigate the major allergen components associated with birch pollen allergy in northern China and elucidate clinical relevance to pollen food allergy syndrome (PFAS). Methods: Fifty-eight patients were recruited for a cross-sectional study and categorized into two groups: PFAS group and non-PFAS group, as well as apple allergy group and non-apple allergy group. The sIgE levels of birch pollen and its components, namely Bet v 1, Bet v 2, Bet v 4, and Bet v 6, were analyzed. Results: Among 58 participants, 44 individuals (75.9%) reported PFAS. 32 out of 44 (72.7%) participants reported apple allergy. Bet v 1 exhibited the highest sensitization rate at 82.8%, followed by Bet v 2 (29.3%) and Bet v 6 (1.7%). The combined sensitization rate for Bet v 1 and/or Bet v 2 was 93.1%. A total of 77.6% of the subjects demonstrated sensitization to single component, while 19.0% exhibited sensitization to two components. The sIgE levels of birch pollen and Bet v 1 were significantly elevated in PFAS group compared to non-PFAS group (p=0.001, p<0.001, respectively), as well as in apple-allergic and non-apple-allergic group (p<0.001, p<0.001, respectively). The optimal cut-off values for birch pollen and Bet v 1 sIgE were determined to be 7.09 kUA/L (with a sensitivity of 84.1% and specificity of 78.6%) and 5.11 kUA/L (with a sensitivity of 75.0% and specificity of 85.7%) when diagnosing PFAS. In terms of apple allergy, the optimal cut-off value were 9.40 kUA/L (with a sensitivity of 81.3% and specificity of 76.9%) and 6.53 kUA/L (with a sensitivity of 84.4% and specificity of 84.6%), respectively. Conclusion: The predominant sensitization pattern is mono-sensitization to Bet v 1, but when considering immunotherapy, Bet v 2 should also be taken into account. Bet v 1 serves as a valuable biomarker for diagnosing PFAS and apple allergy.

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1.

Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01-Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

Comparative analysis of allergen genes and pro-inflammatory factors in pollen and fruit of apple varieties.

Allergy to freshly consumed apple fruits is often associated to pollinosis and manifested as oral allergy syndrome (OAS). The allergenic properties of apple varieties differ greatly, spanning from low allergenic to high allergenic varieties. The knowledge of the genetic determinants for allergenicity has been of great interest in scientific community for several years, but the molecular mechanisms involved are still little understood. Here, factors putatively involved in allergenicity were investigated at biochemical and molecular level in pollen and in fruits of apple varieties differing in their allergenic potential. Among putative sensitizing factors, transglutaminase (TGase) and phospholipase A2 (PLA2) were considered together with reactive oxygen species (ROS) and known apple allergen genes, with particular attention devoted to the Mal d 1 gene family, the most important one in sensitization. We found that the expression of some allergen genes and the activities of TGase, PLA2 and ROS producing enzyme are lower in the hypo-allergenic variety 'Durello di Forlì' in comparison with the high-allergenic genotypes 'Gala' and 'Florina'. These results highlight correlations among allergen expressions, enzymatic activities and apple cultivars; these data underline the possibility that some of them could be used in the future as markers for allergenicity.