Relationship between Phenolic Compounds, Antioxidant Properties, and the Allergenic Protein Mal d 1 in Different Selenium-Biofortified Apple Cultivars (Malus domestica).

Notable parts of the population in Europe suffer from allergies towards apples. To address this health problem, the analysis of the interactions of relevant allergens with other substances such as phenolic compounds is of particular importance. The aim of this study was to evaluate the correlations between the total phenolic content (TPC), polyphenol oxidase (PPO) activity, antioxidant activity (AOA), and the phenolic compound profile and the content of the allergenic protein Mal d 1 in six apple cultivars. It was found that the PPO activity and the content of individual phenolic compounds had an influence on the Mal d 1 content. With regard to the important constituents, flavan-3-ols and phenolic acids, it was found that apples with a higher content of chlorogenic acid and a low content of procyanidin trimers and/or epicatechin had a lower allergenic potential. This is probably based on the reaction of phenolic compounds (when oxidized by the endogenous PPO) with proteins, thus being able to change the conformation of the (allergenic) proteins, which further corresponds to a loss of antibody recognition. When apples were additionally biofortified with selenium, the composition of the apples, with regard to TPC, phenolic profile, AOA, and PPO, was significantly affected. Consequently, this innovative agronomic practice seems to be promising for reducing the allergenic potential of apples.

The Effect of Birch Pollen Immunotherapy on Apple and rMal d 1 Challenges in Adults with Apple Allergy.

BACKGROUND: A proportion of patients allergic to birch pollen are also allergic to pit fruit. The objective of this study was to investigate the effect of immunotherapy with birch pollen on birch-pollen-related apple allergy. METHOD: Patients with birch pollen immunotherapy underwent a skin-prick test with birch pollen, apple and rMal d 1, global assessments and nasal challenges with birch pollen, open food challenge with apple and a double-blind, placebo-controlled test with rMal d 1 at the start of and during the immunotherapy. Measurements of specific IgE in response to Bet v 1 and rMal d 1 and IgG4 in response to Bet v 1 and rMal d 1 took place. RESULTS: Six of eight patients demonstrated an improvement of nasal challenge test results and all patients improved on global assessment during the immunotherapy. The median oral dose of apple required to elicit a reaction increased but was not statistically significant. The patients showed a decrease in skin-prick test values in response to birch pollen (1.05 to 0.36), apple (0.78 to 0.25) and rMal d 1 (0.51 to 0.10) with p-values of 0.04, 0.03 and 0.06, respectively and a decrease of specific IgE in response to Bet v 1 (10.66 kU/L to 5.19 kU/L) and rMal d 1 (0.99 to 0.61 kU/L) with p-values of 0.01 and 0.05, respectively. Only the median specific IgG4 value to Bet v 1 increased from 0.05 to 1.85 mg/L (p-value of 0.02) and not to IgG4 rMal d 1 (0.07 to 0.08 kU/L). CONCLUSION: The beneficial effects of immunotherapy for birch pollen were accompanied by a limited effect on apple allergy.

Dietary Interventions in Pollen-Related Food Allergy.

In practice, it remains unclear what the best dietary approach is in subjects with pollen-related food allergy (PRFA). Our objective was to evaluate the effect of (1) dietary avoidance advice, (2) oral immunotherapy (OIT), (3) (heat) processing, and (4) consumption of hypoallergenic cultivars on frequency, severity, and eliciting dose of pollen-related food allergic reactions. A systematic search was conducted in PubMed, Embase, and Cochrane. All studies performing an in vivo investigation of one of the four interventions in adults with PRFA were included. Each study was assessed for quality and validity. Available data on frequency, severity, and eliciting dose of allergic reactions were extracted. Ten studies matched the eligibility criteria. No studies were retrieved on dietary avoidance advice. Two studies (N = 92) on apple OIT reported that tolerance was induced in 63% and 81% of subjects. Four studies (total N = 116) focused on heat processing. Heating was found to completely eradicate symptoms in 15⁻71% of hazelnut allergic and 46% of celery allergic individuals. Four studies (N = 60) comparing low to high allergenic apple cultivars revealed that Santana (and possibly Elise) apples seemed to cause milder reactions than Golden Delicious. In the awareness that overall level of evidence was low, we conclude that OIT, heat processing, and hypoallergenic cultivars may diminish or completely prevent allergic reactions in some but not all subjects with PRFA.

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.

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.

Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen-allergic patients with apple allergy.

It is still unclear whether allergen-specific immunotherapy (AIT) with birch pollen improves birch pollen-related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen-related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1-homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen-allergic patients with apple allergy. The dose of 1.6 µg induced oral allergy syndromes in 26.4%, 3.2 µg in 15.3%, 6.3 µg in 27.8%, 12.5 µg in 8.3%, 25 µg in 11.1%, and 50 µg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 µg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose-defined sublingual challenge tests in a high number of birch pollen-allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen-related food allergy.

Continuous apple consumption induces oral tolerance in birch-pollen-associated apple allergy.

BACKGROUND: Patients with birch pollen allergy (major allergen: Bet v 1) have often an associated oral allergy syndrome (OAS) to apple, which contains the cross-reactive allergen Mal d 1. As successful birch pollen immunotherapy does not consistently improve apple related OAS symptoms, we evaluated whether regular apple consumption has an effect on OAS and immune parameters of Mal d 1 or Bet v 1 allergy. METHODS: A total of 40 patients with a clear history of birch pollen rhinoconjunctivitis and associated OAS to apple were included in an open, randomized, controlled clinical trial: 27 patients consumed daily defined amount of apple (1-128 g), doubling the amount every two to three weeks, while 13 patients remained untreated. Primary endpoint was the proportion of patients that achieved tolerance to at least 128 g of apple at the end of the study after 8 months. Exploratory endpoints were questionnaire about cross-reactive food and pollen allergy symptoms, conjunctival provocation test with birch pollen and Bet v 1, and in vitro tests (tIgE, sIgE, and IgG4 to Mal d 1 and Bet v 1; basophil activation test with both allergens). RESULTS: Seventeen of 27 patients in active group and none of 13 patients in control group (P = 0.0001) could tolerate a whole apple after the intervention. However, differences in endpoints reflecting systemic immune reactivity did not reach statistical significance. CONCLUSION: In patients with OAS to apple, tolerance can be safely induced with slowly, gradually increasing consumption of apple. However, the observation of a relapse after discounting of apple consumption and absence of immunologic changes suggest that induced tolerance is only transient.

'I've got hay-fever and my mouth is stinging!'.

A case of oral allergy syndrome is presented. Crossreactivity of pollens with some fruit/vegetables causes immediate IgE-mediated symptoms localised to the mouth. Diagnosis is suspected from positive skin prick testing in the presence of a suggestive history. Management is by allergen avoidance. In serious and refractory cases, referral to a regional allergy clinic is recommended.

Birch-apple syndrome treated with birch pollen immunotherapy.

BACKGROUND: The most common pollen-fruit cross-reaction is the birch-apple syndrome. Allergen immunotherapy (IT) is clearly effective for birch allergy, but its efficacy on apple allergy is controversial. We performed a randomized study on patients with birch-apple syndrome to evaluate the outcome of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). METHODS: Forty patients underwent IT with a birch extract (Staloral; Stallergenes, Antony, France), 20 by SCIT and 20 by SLIT. After 1 year of treatment, 15 patients (8 for SCIT and 7 for SLIT) accepted to undergo an oral apple challenge. Measurements of specific IgE to Bet v 1 and Mal d 1 and related allergens Api g 1 and Dau c 1 were obtained in 10 patients, at baseline and after IT. RESULTS: Two of 8 SCIT-treated patients (25%) and 1 of 7 SLIT-treated patients (14.2%) developed complete tolerance to apple. In the remaining patients, an increase in the provocative dose was found in 3 of the SCIT-treated (37.5%) and 2 of the SLIT-treated patients (28.6%). Changes in the levels of specific IgE to Mal d 1 were unrelated to clinical results. CONCLUSIONS: These findings suggest that different doses of birch extract may be needed in different patients to improve the associated apple allergy and that a finer diagnostic work-up in selecting patients with birch-apple syndrome who are candidates to respond to birch pollen IT also concerning apple allergy is required.

Identification of B-cell epitopes of Bet v 1 involved in cross-reactivity with food allergens.

BACKGROUND: The pollen-food syndrome (PFS) is an association of food allergies to fruits, nuts, and vegetables in patients with pollen allergy. Mal d 1, the major apple allergen, is one of the most commonly associated food allergens for birch pollen-allergic patients suffering from PFS. Although the reactions are due to cross-reactive IgE antibodies originally raised against pollen Bet v 1, not every Bet v 1-allergic patient develops clinical reactions towards apple. AIM OF THE STUDY: We speculate that distinct IgE epitopes are responsible for the clinical manifestation of PFS. To test this hypothesis we grafted five Mal d 1 stretches onto Bet v 1. The grafted regions were 7- or 8-amino acids long encompassing amino acids residues previously shown to be crucial for IgE recognition of Bet v 1. METHODS: A Bet v 1-Mal d 1 chimeric protein designated BMC was expressed in Escherichia coli and purified to homogeneity. IgE reactivity of BMC was tested with patients' sera originating from (i) Bet v 1-allergic patients displaying no clinical symptoms upon ingestion of apples; and (ii) Bet v 1-allergic patients displaying allergic symptoms upon ingestion of apples and other Bet v 1-related foods. RESULTS AND CONCLUSION: Compared to birch pollen-allergic individuals, patients suffering from PFS showed significantly higher IgE reactivity with BMC (chimeric protein). The results suggest that the Mal d 1 regions grafted onto the Bet v 1 sequence comprise important IgE epitopes recognized by Bet v 1-allergic patients suffering from allergy to apples.

Search for low-allergenic apple cultivars for birch-pollen-allergic patients: is there a correlation between in vitro assays and patient response?

BACKGROUND: Due to the cross-reactivity between Bet v 1 and proteins present in vegetable foods, birch pollen allergic patients frequently develop allergy to fruits and vegetables, mostly apples. Since many apple cultivars exist some of them might contain sufficiently low amounts of Mal d 1 to be tolerated by most allergic patients. OBJECTIVE: To assess whether apple cultivars containing low amounts of Mal d 1 are better tolerated by apple-allergic patients. METHODS: Mal d 1 content was determined in many apple cultivars by ELISA. Selected cultivars containing high (Golden Delicious) or low (Orim, G 198 and Vienna) amounts of Mal d 1 were compared in apple allergic patients both by SPT and oral challenges. RESULTS: The 3 different apple cultivars induced wheals of similar size in most patients. Upon oral challenges no patient reported the total absence of oral symptoms following the ingestion of either high or low allergenic apples. Golden Delicious and G-198 elicited OAS of similar severity 3/7 cases. The 2 cultivars induced significantly more severe symptoms in 2 cases each. CONCLUSION: Allergy to Mal d 1 is characterized by significant inter-patient variability. Moreover, marked inter-apple and intra-apple variability exists. As a consequence, the amount of Mal d 1 in apples classified as containing low concentrations of allergen may be sufficient to induce both clinical symptoms and skin reactivity in birch pollen-allergic patients. The search for low allergenic apples therefore should be continued and extended to other germplasm accessions, be it cultivars, breeding lines or wild species.

PT with heat-processed apple peel extract to detect LTP hypersensitivity.

BACKGROUND: Allergen-resolved diagnosis of food allergy may be essential in the clinical practice, particularly in patients allergic to foods that may contain both labile and stable allergens. However, presently available diagnostic tests are not useful in this sense. OBJECTIVE: To assess the clinical usefulness of SPT with a heat-processed apple peel extract as an easily available means to detect hypersensitivity to lipid transfer protein (LTP), an extremely stable and potentially hazardous apple allergen. METHODS: Raw and heat-processed (100 degrees C x 10 min) apple peel extract (100 microg/ml) were used to carry out SPT in 23 patients with apple-allergy, 15 of which considered as probably sensitized to labile allergens (Maid 1, Mal d 4) and 8 to stable allergens (Mal d 3, LTP), respectively on the basis of the presence/absence of IgE reactivity to birch pollen. IgE reactivity to the heat-processed apple peel extract was further analyzed by immunoblot. RESULTS: Altogether SPT with raw apple extract scored positive in 20/23 (87%) patients, including 12/15 patients considered as probably sensitized to labile allergens and 8/8 patients considered as probably sensitized to LTP. In contrast, the heat-processed apple extract induced a wheal-and-flare reaction only in the 8 (100%) presumptive LTP reactors. Immunoblot analysis showed IgE reactivity to a 10 kDa protein (LTP) in heat-processed apple extract. CONCLUSION: The heat-resistance of stable apple allergens like LTP can be usefully exploited to prepare extracts for allergen-resolved diagnosis in-vivo.

Food allergy to apple and specific immunotherapy with birch pollen.

Conflicting results concerning the effect of specific pollen immunotherapy (SIT) on allergy to plant foods have been reported. The aim of this study was to investigate the effect of SIT using a birch pollen extract on food allergy with focus on allergy to apple. Seventy-four birch pollen-allergic patients were included in a double-blind, double-dummy, and placebo-controlled comparison of sublingual-swallow (SLIT) and subcutaneous (SCIT) administration of a birch pollen extract. Sixty-nine percent of these patients reported allergy to apple. The clinical reactivity to apple was evaluated by open oral challenges with fresh apple and a questionnaire. The immunoglobulin E (IgE)-reactivity was assessed by skin prick test (SPT), specific IgE, and leukocyte histamine release (HR). Forty patients were included in the final evaluation of the effect of SIT. The challenges were positive in 9 (SCIT), 6 (SLIT), and 8 (placebo) patients after treatment compared to 10, 4, and 10 patients, respectively, before SIT. The symptom scores to apple during challenges decreased in all groups, but only significantly in the placebo group (p = 0.03). As evaluated by the questionnaire, the severity of food allergy in general did not change and there were no differences between the groups. In spite of a significant effect on seasonal hay fever symptoms and use of medication and decrease in IgE-reactivity, SIT was not accompanied by a significant decrease in the severity of allergy to apple compared to placebo. Therefore, oral allergy syndrome (OAS) to apple should not be considered as a main criterion for selecting patients for birch pollen immunotherapy at present.

Effect of tree pollen specific, subcutaneous immunotherapy on the oral allergy syndrome to apple and hazelnut.

BACKGROUND: The efficacy of specific immunotherapy (SIT) in pollen allergy is well established. However, its effect on pollen associated food allergy particularly the oral allergy syndrome (OAS) is not definitely ascertained. OBJECTIVE: The purpose of this controlled prospective study was to investigate whether SIT with tree pollen, mainly birch, has an effect on OAS induced by apple or hazelnut in birch pollen-allergic individuals. METHODS: Twenty-seven birch pollen-allergic subjects with OAS induced by apple or hazelnut underwent open oral provocation tests (OPT) with increasing doses (1 to 128 g) of fresh apple or ground hazelnut 1 year apart. Fifteen of 27 subjects were treated with SIT and 12 were not. Skin-prick test with birch pollen, apple and hazelnut, and specific serum IgE, IgG and IgG4 to rBet v 1, apple and hazelnut were determined. RESULTS: Thirteen of 15 (87%) SIT-treated subjects could eat significantly (P <0.001) more of apple or hazelnut without any symptoms/signs. The average tolerated quantity increased from 12.6 to 32.6 g apple after 1 year in this group. In contrast, only one of 12 (8%) individuals without SIT was able to consume a higher amount without symptoms. On evaluating laboratory parameters, only IgG4 antibodies to rBet v 1 were found to be significantly (P <0.01) increased in the SIT-treated group after 1 year. CONCLUSIONS: The study shows that SIT with extracts containing birch pollen has a positive impact on OAS to apple or hazelnut in birch pollen-allergic individuals. In spite of this outcome, the amount of apple/hazelnut tolerated is still small. Thus, the effect of SIT on the patients' management of OAS remains limited.

Reduction of the pH-values of whole saliva after the intake of apple juice containing beverages in children and adults.

PURPOSE: The aim of the present study was to compare changes in salivary pH after intake of apple juice and its various dilutions with mineral water, in children and adults. MATERIALS AND METHODS: 15 children (mean age 4.4+/-0.9 years), and 15 adults (mean age 30+/-2.4 years) participated in this study. Carbonated mineral water, apple juice and its various dilutions with carbonated mineral water were tested. The pH-value of unstimulated whole saliva was measured at the same time of day. The pH-value was measured again immediately after intake of a beverage, and 5, 10, 15 and 25 min later. The flow rate and buffering capacity of stimulated saliva, and the buffering capacity, calcium and phosphorus contents of the beverages were measured. The statistical evaluation of the data was performed employing the Wilcoxon test for paired samples. RESULTS: The mean base value of the pH of whole saliva was 7.0+/-0.2 (children), and 6.8+/-0.3 (adults). The differences in the flow rates of stimulated saliva in children and adults were statistically significant (p = 0.0003). The intake of mineral water led to a statistically significant rise in the salivary pH-value (p < 0.05). Undiluted and diluted apple juice caused a reduction in the salivary pH-values. Within the first 10 min the changes of pH in saliva only differed significantly between mineral water and the other beverages (p < 0.01). In the pH range of 5.8 - 7.0, mineral water had a weak buffering capacity, while undiluted apple juice had a high buffering capacity. CONCLUSIONS: With regard to dental and general health, only mineral water can be recommended for children.