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BackgroundLimited is known on the profiles of apple allergy in China.ObjectiveTo explore the clinical significance of apple allergen components in northern China.MethodsThis 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.ResultsThe 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

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.

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.

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. [ABSTRACT FROM AUTHOR]

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'. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The consumption of apple fruits can induce allergic reactions. The major allergen responsible for these allergic reactions in Central Europe and North America is the apple allergen Mal d 1, the synthesis of which depends on the apple cultivar and increases during storage time. Several studies have investigated the impact of the cultivar and storage conditions on the Mal d 1 content in apple fruits. However, there are few data available on the impact of 1-methylcyclopropene (1-MCP) on Mal d 1 synthesis during long-term storage. In this study, the Mal d 1 content of seven different apple cultivars was analyzed. Half of the fruits were treated with 1-MCP (Smart Fresh™, Agro Fresh Inc. Spring House, PA, USA). After treatment, the fruits were stored for 12, 20, 28 and 36 weeks in a cold chamber at 2 °C as well as for 12, 20, 28, 36 and 44 weeks under controlled atmosphere (CA-storage, 1.5% CO2, 1.5% O2, 2 °C). The Mal d 1 contents of all apple cultivars increased during storage. With the exception of ‘Boskoop’, Mal d 1 contents of all of the fruits treated with 1-MCP were significantly lower than those of the fruits in the untreated control group. [ABSTRACT FROM AUTHOR]

Abstract: The most important apple allergen in Central Europe and North America is Mal d 1. Apples are a very important source of secondary plant metabolites like polyphenols in human nutrition. It is known that oxidised phenols can bind proteins. These irreversible bindings can lead to a reduced allergenicity. The most important phenols in apple are epicatechin, catechin and their polymeric structures, which have been identified as substrates of the polyphenoloxidase (PPO). The aim of this study was to analyse the influence of naturally occurring catechin and epicatechin contents in apple on the allergenicity of apple fruits. Fruits of the cultivars ‘Elstar’, ‘Diwa’ and ‘Boskoop’ were harvested and stored for 8 and 12weeks in a cold-chamber at 2°C. Mal d 1-, catechin-, epicatechin- and total phenol content as well as the activity of PPO were determined. Correlation analysis showed that naturally occurring catechin as well as epicatechin has no impact on the Mal d 1 content of the tested cultivars: correlation coefficient ranged from −0.203 to 0.501 for the correlation between Mal d 1 and catechin. The results further indicated that the activity of PPO is more important than the content of total phenols to reduce the Mal d 1 level. If there is a high PPO activity, Mal d 1 could be reduced even if the total phenol concentration is low. [Copyright &y& Elsevier]

Background: A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars 'Florina' and 'Gala'. Results: We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent. Conclusion: The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy. [ABSTRACT FROM AUTHOR]

To evaluate differences in skin prick test response to apple cultivars in patients with apple allergy, 19 patients (10 adults and 9 children) underwent prick-to-prick skin prick test with eleven commercial and noncommercial apple cultivars, and evaluation of specific IgE to apple and recombinant apple allergens Mal d 1 and Mal d 3. The results show that different reactions might be evoked in a single patient by different apple cultivars and also separately for the peel and the pulp of a single cultivar. The cultivars were ranked according to their allergenicity level, being Jonathan, Jonagold, Golden Delicious and Fiesta the most allergenic when considering the pulp and Gala, Fiesta and Golden Delicious for the peel. Further investigations are needed to clarify if a single patient can be allergic only to well-defined apple cultivars and which allergometric tests are necessary to ascertain this. [ABSTRACT FROM AUTHOR]

Background: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. Methods:A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. Results: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in gE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Apple allergy is a well-characterized fruit allergies linked to pollinosis. • Concerning type and amount of allergens apple cultivars show broad biodiversity. • Five Mal d 1 isoallergens were tested for the first time against allergic patient's sera. • Besides apple varieties, differences among isoallergens and patients were found. Apple-related allergy is increasing among European population. Avoidance of apple fruit consumption is the only strategy for sensitized people, with a subsequent shortage of healthy nutrients in the diet. Four different protein families, Mal d 1 to 4, determine the allergy to apple causing syndromes known as oral allergy syndrome (OAS) or gastro-intestinal/generalized syndrome (GS) in sensitized patients. With the aim to highlight the dissimilarities in the immunological response inside the Mal d 1 family, recognized as main determinant for OAS, single purified allergens were tested against sera of paediatric and adult patients suffering for both syndromes. Here, we found that Mal d 1 isoallergens showed a remarkable different allergenicity potential, thus indicating an individual pattern in each patient and a diverse sensitization between age groups. Moreover, two different allergens, Mald 2.01 and Mal d 4.01, were analyzed with the above sera. In addition to patients and allergens variability, differences in amount and localization of the allergens, detected by patient's sera, were revealed among hypo- and hyper allergenic apple varieties, confirming the complexity of this food allergy. [ABSTRACT FROM AUTHOR]