La pomme

variété/cultivar

Mela campanina	33	16
Ambrosia	14	27
DL35 bio	14	29
Fuji	13	51
Golden	12		5,5
G198	3,5		5
G185	1,5	14
G362	1,5	9
Orim	1		2,5

variété/cultivar

Starking	55
Pink lady	27	8
Fuji	23	9	50
Gala	13	3
Reineta parda	13
Golden	12	12	60
Granny	10	17	100
Canada		2
Topaz			165
Santana			70
Ecolette			30

[1] - Ebner C, Birkner T, Valenta R, Rumpold H, Breitenbach M, Scheiner O, et al. Common epitopes of birch pollen and apples--studies by western and northern blot . J Allergy Clin Immunol 1991;88:588-594

Eighty-three sera from patients with birch-pollen allergy were investigated for IgE antibodies against apple allergens by means of immunoblotting. In immunoblots, 81 patients (97.6%) exhibited IgE directed against the major allergen of birch, Bet v I (17 kd), and these patients also demonstrated IgE binding to apple allergens in the molecular weight range 17 to 18 kd. Inhibition studies by preincubation of sera with birch-pollen extract led to complete blocking of IgE binding to this 17 to 18 kd protein, whereas preincubation with apple extract could not diminish IgE binding to Bet V I. Furthermore, a 17 kd protein in apple extract could be detected by immunoblotting with a Bet v I-specific monoclonal antibody. Northern blotting with a Bet v I cDNA clone as a probe revealed cross-hybridization of birch and apple allergen coding nucleic acids under conditions of high stringency, suggesting significant homology of the nucleic acid level. Our results support the concept that antigens in birch pollen and apples share allergenic epitopes leading to IgE cross-reactivities that may cause clinical manifestations when a special threshold level of specific IgE antibodies is reached

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[2] - Osterballe M, Hansen TK, Mortz CG, Bindslev-Jensen C. The clinical relevance of sensitization to pollen-related fruits and vegetables in unselected pollen-sensitized adults. Allergy 2005;60:218-225

BACKGROUND: Previous studies have described cross-reactivity between fresh fruits, vegetables and pollen. However, no data demonstrates the clinical relevance of sensitization to pollen-related fruits and vegetables in unselected pollen-sensitized adults with and without symptoms in the pollen season . OBJECTIVE: The aim of this study was to estimate the clinical relevance of sensitization to pollen-related fruits and vegetables in unselected pollen-sensitized adults and to examine the diagnostic value of skin-prick test (SPT), histamine release and specific IgE compared with the outcome of oral challenge . METHODS: In total, 936 unselected adults (female : male 479 : 457, median age 33.7 years) were examined for pollen sensitization and clinical cross-reactivity with pollen-related fruits and vegetables by questionnaire, SPT, histamine release, specific IgE and oral challenge . RESULTS: The prevalence of pollen sensitization was 23.8% (n = 223). The probability of a clinical reaction to pollen-related foods in the respective pollen-sensitized groups was: 24% (birch), 4% (grass), 10% (mugwort), 35% (birch + grass), 8% (grass + mugwort) and 52% (birch + grass + mugwort). The odds ratio of a clinical reaction to pollen-related fruits and vegetables in symptomatic pollen-sensitized adults was as high as four times (birch + grass) the odds ratio of a clinical reaction in asymptomatic pollen-sensitized adults . CONCLUSION: This study not only demonstrates a high prevalence of clinical reactions to fruits and vegetables in pollen-sensitized adults, but also a discrepancy between the prevalence of sensitization to fruits and vegetables and the clinical relevance in different pollen-sensitized groups with symptoms in the pollen season as a significant factor.

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[3] - Cuesta-Herranz J, Lazaro M, Figueredo E, Igea JM, Umpierrez A, De Las Heras M. Allergy to plant-derived fresh foods in a birch- and ragweed-free area. Clin Exp Allergy 2000;30:1411-1416

BACKGROUND: Allergy to plant-derived fresh foods has often been reported in geographical areas where birch or ragweed pollens are frequent and has been attributed to cross-reactivity to pollens. OBJECTIVE: The aim of this study has been to evaluate allergy to plant-derived fresh foods among pollen-allergic patients from a birch and ragweed-free area. METHODS: Ninety-five pollen-allergic patients took part in the study. The study consisted of a questionnaire, skin prick tests and challenge tests. Pollen skin tests to five grasses, eight trees and seven weeds were performed in duplicate. Prick tests (prick by prick) and challenge tests were carried out with the fresh foods. RESULTS: Most patients allergic to pollens were sensitized to grass (Lolium and Phleum; 97.9%), followed by tree (Olea; 82.1%) and weed pollens (Plantago; 64.2%). 35 of the 95 pollen-allergic patients had positive skin test responses to some plant-derived fresh foods, the highest percentage corresponding to several fruits in the Rosaceae family (peach and pear, 26.3%), followed by Cucurbitacea fruits (melon, 13.7%). The 21. 05% of the pollen-allergic patients were allergic to some type of plant-derived fresh food. Peach was the plant-derived fresh food which most frequently elicited allergy symptoms (12.6%), followed by melon (7.36%). The cluster of positive responses to Rosaceae fruits was higher for skin testing than for challenge testing. CONCLUSION: Peach was the most important allergy provoking fruit in a birch and ragweed free-area where apples were consumed at a rate of two times more than peaches and the patients allergic to pollen were principally sensitized to grass pollens.

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[4] - de Groot H, de Jong NW, Vuijk MH, Gerth van Wijk R. Birch pollinosis and atopy caused by apple, peach, and hazelnut: comparison of three extraction procedures with two apple strains. Allergy 1996;51:712-718

This study aimed, first, to study the prevalence in The Netherlands of atopy caused by apple, peach, and hazelnut in patients with tree pollinosis, and, second, to compare three extraction procedures for skin prick testing with two different apple strains. Skin prick tests and RAST were performed on 79 consecutive patients with tree pollinosis, visiting the department of allergology during spring 1995. In skin prick tests, we used three different extracts (juice, freeze-dried extract, and low-temperature acetone powder extract) of two apple strains, Golden Delicious and Granny Smith. Case histories for apple, peach, and hazelnut were positive in 35 (44.3%), 23 (29%), and 35 (44.3%) patients, respectively. More than two-thirds of the patients had symptoms characteristic of oral allergy syndrome. Skin prick tests for apple, peach, and hazelnut were positive in 51 (64.6%), 61 (77.2%), and 71 (89.9%) patients, respectively. Granny Smith showed more positive skin reactions and a better agreement with clinical history than Golden Delicious, and juice was superior to the two other extraction procedures for both apple strains. RAST for apple, peach, and hazelnut was positive in 53 (68.8%), 13 (16.9%), and 31 (40.3%) patients, respectively. Concordance between skin prick test and case history was found in 77%, 52%, and 54%, for apple, peach, and hazelnut, respectively. We found a high percentage of concurrence of clinical allergy to birch pollen and apple, peach, and hazelnut, confirmed by both skin prick testing and RAST. Approximately half of these patients had symptoms (especially oral allergy syndrome) after eating these products. We also found an easy extraction procedure (juice extract) suitable for apple skin prick testing, superior even to freeze-dried extraction or the low-temperature acetone powder technique.

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[5] - Metz-Favre C, Birba E, Metivier AC, de Blay F, Pauli G. Intérêt du dosage des IgE vis-à-vis de l'allergène recombinant rBet v 1 dans la prise en charge de la pollinose printanière. Rev Fr Allergol Immunol Clin 2005;45:584-589

Nous avons évalué le dosage des IgE spécifiques anti-rBet v 1 en tant que nouvel outil diagnostique dans la prise en charge de la pollinose printanière. Méthodes. - Cent dix-sept dossiers de patients consultant en allergologie ont été sélectionnés sur la demande d'IgE anti-rBet v 1. Les motifs de consultation étaient variés. Des dosages d'IgE anti-rBet v 2 et antipollen de bouleau (t3) étaient présents respectivement dans 82 et 28 dossiers. Le diagnostic de pollinose au bouleau était établi sur une histoire clinique évocatrice associée à un test cutané positif à l'extrait de pollen de bouleau. La présence d'un syndrome oral a été recherchée chez 105 patients. L'association d'un asthme a été considérée comme un facteur de gravité de la pollinose. Résultats. - Dans la population étudiée, la sensibilité, la spécificité, la VPP et la VPN du dosage d'IgE anti-rBet v 1 sont excellentes (respectivement de 91, 82, 92 et 93 %). Dans le groupe de patients ayant eu également des dosages d'IgE anti-t3, la sensibilité et la spécificité des IgE anti-rBet v 1 étaient supérieures à celles des IgE anti-t3. Lorsque les dosages d'IgE anti-rBet v 2 étaient effectués concomitamment, ils étaient positifs dans 19,5 % des cas avec une médiane de 2,46 KU/l. Le syndrome oral était plus fréquemment associé chez les patients ayant des taux d'IgE spécifiques anti-rBet v 1 supérieurs à 50 KU/l. Le dosage des IgE spécifiques rBet v 1 n'était en revanche pas un élément prédictif de l'association d'un asthme à la pollinose. Conclusion. - Ce dosage permet de définir en partie le profil de sensibilisation des patients et de guider la désensibilisation.

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[6] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[7] - Lahti A, Bjorksten F, Hannuksela M. Allergy to birch pollen and apple, and cross-reactivity of the allergens studied with the RAST. Allergy 1980;35:297-300

Apple-pulp allergens are unstable, but fresh material can be used for skin testing. In this study stable allergens, probably of a protein nature, were extracted from apple seed and used in radioallergosorbent tests (RAST). Apple-seed allergens were shown by RAST inhibition to cross-react with birch pollen allergen(s). Cross-reactivity may explain the frequent co-occurrence of birch pollen and apple allergy. Approximately 50% of patients in a group with immediate hypersensitivity to birch pollen were also sensitive to apple as judged by a history of symptoms, skin tests with apple-pulp and apple-seed preparations, and apple-seed RAST.

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[8] - Dreborg S, Foucard T. Allergy to apple, carrot and potato in children with birch pollen allergy. Allergy 1983;38:167-172

Skin sensitivity to apple, carrot and potato, clinically related to birch pollinosis was investigated. Different skin test techniques using fresh fruit were compared. A simple prick test (SPT) technique with a lancet piercing the apple peel just before pricking the skin was shown to be the most practical and to give reproducible results. The allergenic activity in apple was found to be heat labile and deteriorated during storage at room temperature. Apple, carrot, potato, hazelnut and birch reactivity was transferable in Prausnitz-Kustner test like IgE antibodies. SPT reactivity to fresh material from apple, carrot and potato was investigated in 174 children of whom 128 suffered from pollen allergy. Positive SPT results were obtained almost exclusively in children who were SPT positive to a birch pollen extract. Children who noticed clinical symptoms when eating apple, raw carrot or potato were found to have a significantly larger SPT reaction than children with a negative history.

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[9] - Rossi RE, Monasterolo G, Operti D, Corsi M. Evaluation of recombinant allergens Bet v 1 and Bet v 2 (profilin) by Pharmacia CAP system in patients with pollen-related allergy to birch and apple. Allergy 1996;51:940-945

Sixty-five patients presenting either rhinoconjunctivitis or asthma and sensitized to pollens of trees of the order Fagales were studied by the Pharmacia CAP system in order to assess specific IgE for the important birch pollen allergens Bet v 1 and Bet v 2. All 65 subjects reacted to at least one of the recombinant birch allergens: 43% to Bet v 1, 30.7% to Bet v 2, and 26% to both. Patients monosensitized to birch did not react to Bet v 2. Of patients with a history of oral allergy syndrome after eating apples, 16/28 (57%) reacted to Bet v 1; among 20 polysensitized subjects presenting oral allergy syndrome after consumption of apple, four reacted to Bet v 2 (20%). Among patients with IgE against both recombinant allergens, six (35.30%) presented symptoms of allergy after eating apples Our results indicate that sensitization to Bet v 1 is specific for birch and apple allergies, whereas sensitization to Bet v 2 is common in polysensitized patients.

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[10] - Patriarca S, Voltolini S, Navone R, Martini S, Montanari C, Negrini A, et al. Biochemical and immunochemical characterization of hop-hornbeam (Ostrya Carpinifolia Scop.) pollen - A new allergenic pollen from the Corylaceae family. Aerobiologia 2000;16:255-260

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[11] - Kleine-Tebbe J, Wangorsch A, Vogel L, Crowell DN, Haustein UF, Vieths S. Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1-related PR-10 protein in soybean, SAM22. J Allergy Clin Immunol 2002;110:797-804

BACKGROUND: Anaphylactic reactions to soy products have been attributed to stable class 1 food allergens . OBJECTIVE: IgE- mediated reactions to a soy-containing dietary food product in patients allergic to birch pollen were investigated . METHODS: Detailed case histories were taken from 20 patients. Their sera were analyzed for IgE (UniCAP) specific for birch, grass, mugwort, the recombinant birch allergens rBet v 1 and rBet v2, and soy protein. Extracts from birch pollen, soy isolate, rBet v 1, and the recombinant PR-10 soy protein rSAM22 were coupled to paper disks or nitrocellulose for IgE measurements (enzyme allergosorbent test) or Western blot analysis. Enzyme allergosorbent testing, Western blot inhibition, and histamine release studies were performed with the same allergens . RESULTS: Most patients (17/20) experienced facial, oropharyngeal, and/or systemic allergic symptoms within 20 minutes after ingesting the soy product for the first time. Birch pollen allergy (16/20) was common, along with oral allergy syndrome to apple (12/20) or hazelnut (11/20). IgE levels to birch and Bet v 1 but not to other inhalants were high in 18 of 20 patients. Significant IgE binding to rSAM22 occurred in 17 of 20 patients. Blot experiments with the soy isolate revealed IgE-binding bands at 17 kd (15/20), 22 kd (1/20), and 35 to 38 kd (2/20); the former was inhibited by preincubation of the sera with rBet v 1 or rSAM22. Birch extract and soy isolate, rBet v 1, and rSAM22 induced dose-dependent histamine release in the nanomolar range . CONCLUSION: Immediate-type allergic symptoms in patients with birch pollen allergy after ingestion of soy protein-containing food items can result from cross-reactivity of Bet v 1 -specific IgE to homologous pathogenesis-related proteins, particularly the PR-10 protein SAM22.

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[12] - Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A, et al. Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004;113:148-154

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[13] - Mittag D, Vieths S, Vogel L, Wagner-Loew D, Starke A, Hunziker P, et al. Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1. Clin Exp Allergy 2005;35:1049-1055

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1 . METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS) . RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress . CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.

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[14] - Reuter A, Lidholm J, Andersson K, Ostling J, Lundberg M, Scheurer S, et al. A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe. Clin Exp Allergy 2006;36:815-823

BACKGROUND: Food allergy to cherry occurs throughout Europe, typically with restricted oral reactions in the central and northern parts but with frequent systemic reactions in the Mediterranean region. Previous studies have demonstrated insufficient sensitivity of commercially available cherry extract reagents in the diagnosis of cherry allergy . OBJECTIVE: To assess the diagnostic performance of specific IgE tests based on recombinant cherry allergens in comparison with an extract-based assay and to skin prick test (SPT). A secondary objective was to analyse the frequency of systemic reactions in cherry-allergic subjects across Europe, including the largest population of LTP-sensitized subjects from central Europe studied to date . METHODS: A total of 186 subjects from central Europe and Spain were studied. Serum IgE was analysed with ImmunoCAP tests carrying rPru av 1, 3 and 4, combined and separately, and cherry extract . RESULTS: Among the central European cherry allergics, the mix of rPru av 1, 3 and 4 had a sensitivity of 95%, compared with 65% for cherry extract, and the IgE binding capacity of the recombinant mix was considerably higher. The sensitivity of the two tests was more comparable in the Spanish population, 95% and 86%, respectively. The recombinant allergen ImmunoCAP equalled SPT in terms of sensitivity and specificity. Consistent with previous reports, major geographic differences in sensitization pattern and prevalence of systemic reactions were found. A significantly higher rate of systemic reactions was found in Spanish patients sensitized to Pru av 3 whereas German patients sensitized to LTP only had oral allergy syndrome . CONCLUSIONS: The recombinant cherry allergen ImmunoCAP is a highly sensitive diagnostic tool, clearly superior to any diagnostic method based on cherry extract. Three cherry allergens are sufficient for detecting sensitization in 95% of cherry-allergic subjects. Systemic reactions are common in LTP-sensitized individuals but seem to require at least one additional causative factor.

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[15] - Pauli G, Bessot JC, Dietemann-Molard A, Braun PA, Thierry R. Celery sensitivity: clinical and immunological correlations with pollen allergy. Clin Allergy 1985;15:273-279

The authors studied twenty patients with celery allergy and concomitant hypersensitivity to certain pollens (mugwort, birch). The specific symptoms induced by eating celery were attacks of urticaria and angio oedema (seventeen out of twenty) respiratory complaints (eight out of twenty), systemic anaphylaxis with vascular collapse (three out of twenty). A strong association between clinical reactions to celery and mugwort sensitization, and to a lesser degree between celery allergy and birch pollen sensitization was established. Celery allergy is mediated by IgE antibodies and can be easily diagnosed by cutaneous tests using fresh material and/or by adequate RAST test. RAST inhibitions performed on individual sera suggest the existence of common antigens in celery and mugwort, and in celery and birch pollen. However, the exact nature of these common antigens has not yet been determined.

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[16] - Asero R. Relevance of pollen-specific IgE levels to the development of Apiaceae hypersensitivity in patients with birch pollen allergy. Allergy 1997;52:560-564

A large clinical/serologic study was carried out to determine the prevalence of Apiaceae (carrot, celery, and fennel) hypersensitivity in patients with birch pollen allergy, and to investigate its relationship with apple and hazelnut allergy and with birch pollen-specific IgE levels. A total of 196 birch pollen-hypersensitive patients with oral allergy syndrome (OAS) caused by different vegetable foods were examined in the cross-sectional part of the study. Of this total, 195 patients had apple and/or hazelnut allergy, and 103 had Apiaceae sensitivity; only one patient had Apiaceae allergy alone. Apiaceae-positive patients showed significantly higher birch pollen-specific IgE levels than negative ones (median 13 vs 7 AU/ml; P<0.0001). The prospective part of the study was performed on 103 birch pollen-hypersensitive patients who were OAS-free at the time of the first visit and were periodically followed-up for OAS. Patients who developed Apiaceae sensitivity showed much higher birch-specific IgE levels than patients who developed apple/hazelnut allergy only (median 15.5 vs 8.5 AU/ml; P<0.05), whereas those who remained OAS-free showed the lowest specific IgE levels (median 5 AU/ml). This study suggests that most Apiaceae determinants cross-react with apple or hazelnut determinants, whereas only some apple or hazelnut determinants cross-react with apiaceae-allergenic determinants; moreover, it shows that birch-specific IgE levels heavily influence the onset of OAS as a whole, and probably play a critical role in the development of allergies to distinct vegetable foods as well

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[17] - Asero R, Mistrello G, Roncarolo D, Amato S, Zanoni D, Barocci F, et al. Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. J Allergy Clin Immunol 2003;112:427-432

BACKGROUND: A proper classification of patients allergic to plant-derived foods is of pivotal importance because the clinical features of allergic reactions to fruits and vegetables depend on the nature and characteristics of proteins responsible for sensitization. However, in normal clinical settings this is presently impossible . OBJECTIVE: We sought to detect clinical markers of sensitization to profilin . METHODS: Seventy-one patients allergic to fruits and vegetables but not sensitized to lipid transfer protein or natural rubber latex were studied. Food allergy was ascertained on the basis of clinical history and positive skin prick test responses with fresh foods, commercial extracts, or both. Allergies to foods that had caused less than 2 adverse reactions were confirmed by means of open oral challenge. IgE reactivity to rBet v 1/rBet v 2 and to natural Phleum species profilin were detected. Moreover, IgE to the 30- to 40-kd and 60- to 90-kd birch pollen-enriched fractions, which also can be involved in cross-reactivity phenomena, were measured in sera from 52 patients by means of ELISA . RESULTS: On the basis of in vitro tests, 24, 18, and 25 patients turned out to be sensitized to Bet v 1, Bet v 2, or both, respectively. Four patients had negative test results for both allergens. Hypersensitivity to Bet v 2 was strongly associated with clinical allergy to citrus fruits (39% in patients monosensitized to Bet v 2 vs 4% in patients monosensitized to Bet v 1, P <.025), melon or watermelon (67% vs 0%, P <.001), banana (66% vs 8%, P <.001), and tomato (33% vs 0%, P <.05), whereas Bet v 1 sensitivity was associated with clinical allergy to apple (100% vs 39%, P <.001) and hazelnut (56% vs 0%, P <.001). The sensitivity of a history of allergy to gourd fruits, citrus fruits, tomato, banana, or a combination thereof as a means to detect profilin-hypersensitive patients was 85% (41/48). The specificity of an allergy to any of these fruits exceeded 85%, with positive predictive values ranging between 68% and 91% . CONCLUSION: In clinical settings in which laboratory investigations are not easily accessible, allergy to melon, watermelon, citrus fruits, tomato, and banana can be used as a marker of profilin hypersensitivity once a sensitization to natural rubber latex and lipid transfer protein is ruled out.

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[18] - Asero R, Mistrello G, Roncarolo D, Amato S, Zanoni D, Barocci F, et al. Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. J Allergy Clin Immunol 2003;112:427-432

BACKGROUND: A proper classification of patients allergic to plant-derived foods is of pivotal importance because the clinical features of allergic reactions to fruits and vegetables depend on the nature and characteristics of proteins responsible for sensitization. However, in normal clinical settings this is presently impossible . OBJECTIVE: We sought to detect clinical markers of sensitization to profilin . METHODS: Seventy-one patients allergic to fruits and vegetables but not sensitized to lipid transfer protein or natural rubber latex were studied. Food allergy was ascertained on the basis of clinical history and positive skin prick test responses with fresh foods, commercial extracts, or both. Allergies to foods that had caused less than 2 adverse reactions were confirmed by means of open oral challenge. IgE reactivity to rBet v 1/rBet v 2 and to natural Phleum species profilin were detected. Moreover, IgE to the 30- to 40-kd and 60- to 90-kd birch pollen-enriched fractions, which also can be involved in cross-reactivity phenomena, were measured in sera from 52 patients by means of ELISA . RESULTS: On the basis of in vitro tests, 24, 18, and 25 patients turned out to be sensitized to Bet v 1, Bet v 2, or both, respectively. Four patients had negative test results for both allergens. Hypersensitivity to Bet v 2 was strongly associated with clinical allergy to citrus fruits (39% in patients monosensitized to Bet v 2 vs 4% in patients monosensitized to Bet v 1, P <.025), melon or watermelon (67% vs 0%, P <.001), banana (66% vs 8%, P <.001), and tomato (33% vs 0%, P <.05), whereas Bet v 1 sensitivity was associated with clinical allergy to apple (100% vs 39%, P <.001) and hazelnut (56% vs 0%, P <.001). The sensitivity of a history of allergy to gourd fruits, citrus fruits, tomato, banana, or a combination thereof as a means to detect profilin-hypersensitive patients was 85% (41/48). The specificity of an allergy to any of these fruits exceeded 85%, with positive predictive values ranging between 68% and 91% . CONCLUSION: In clinical settings in which laboratory investigations are not easily accessible, allergy to melon, watermelon, citrus fruits, tomato, and banana can be used as a marker of profilin hypersensitivity once a sensitization to natural rubber latex and lipid transfer protein is ruled out.

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[19] - Reuter A, Lidholm J, Andersson K, Ostling J, Lundberg M, Scheurer S, et al. A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe. Clin Exp Allergy 2006;36:815-823

BACKGROUND: Food allergy to cherry occurs throughout Europe, typically with restricted oral reactions in the central and northern parts but with frequent systemic reactions in the Mediterranean region. Previous studies have demonstrated insufficient sensitivity of commercially available cherry extract reagents in the diagnosis of cherry allergy . OBJECTIVE: To assess the diagnostic performance of specific IgE tests based on recombinant cherry allergens in comparison with an extract-based assay and to skin prick test (SPT). A secondary objective was to analyse the frequency of systemic reactions in cherry-allergic subjects across Europe, including the largest population of LTP-sensitized subjects from central Europe studied to date . METHODS: A total of 186 subjects from central Europe and Spain were studied. Serum IgE was analysed with ImmunoCAP tests carrying rPru av 1, 3 and 4, combined and separately, and cherry extract . RESULTS: Among the central European cherry allergics, the mix of rPru av 1, 3 and 4 had a sensitivity of 95%, compared with 65% for cherry extract, and the IgE binding capacity of the recombinant mix was considerably higher. The sensitivity of the two tests was more comparable in the Spanish population, 95% and 86%, respectively. The recombinant allergen ImmunoCAP equalled SPT in terms of sensitivity and specificity. Consistent with previous reports, major geographic differences in sensitization pattern and prevalence of systemic reactions were found. A significantly higher rate of systemic reactions was found in Spanish patients sensitized to Pru av 3 whereas German patients sensitized to LTP only had oral allergy syndrome . CONCLUSIONS: The recombinant cherry allergen ImmunoCAP is a highly sensitive diagnostic tool, clearly superior to any diagnostic method based on cherry extract. Three cherry allergens are sufficient for detecting sensitization in 95% of cherry-allergic subjects. Systemic reactions are common in LTP-sensitized individuals but seem to require at least one additional causative factor.

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[20] - Akkerdaas J, Fernandez-Rivas M, Zuidmeer L, Hefle S, Aalberse R, van Ree R. IgE binding profiles to several food LTPs of Spanish patients with apple ingestion related symptoms. Allergy Clin Immunol Int 2005;17(Suppl. 1):338-339

Background Fruit allergic individuals from the Mediterranean area are predominantly sensitized by the non-specific lipid transfer protein (nsLTP). It seems that peach LTP is the most likely sensitizing allergen, however other factors (pollen related, genetic) can not be excluded yet. Objective It was our aim to identify IgE binding profiles to different natural (n) and recombinant (r) purified LTPs and to study the pair wise correlation between amino acid sequence identity and LTP specific IgE titers. Methods Serum and clinical data (after ingestion of 6 different foods) was obtained from Spanish apple allergic patients (n=19), as was proven by SPT/ DBPCFC. Seven different LTPs (rApple, rPeach, nGrape, rStrawberry, nSunflower, nHazelnut and nMaize, also analyzed for sequence homology) were applied in RAST to determine specific-IgE titers. Results Clinical symptoms after apple ingestion ranged from no- to severe systemic reactions like anaphylaxis. LTP Sequence homologies ranged from 81% (apple/peach) to 44% (hazelnut/maize). Highest correlation observed for IgE binding:0.989 (apple:peach). Lowest correlation: 0.228 (maize/grape). RAST highest/lowest mean score: apple 6.4/maize 0.4 IU/ml respectively. Conclusion Since most patients were selected on having a true apple allergy and the amino acid sequence homology between apple and peach LTP is 81%, an IgE binding-correlation of 0.989 was to be expected. Elucidation of IgE crossreactivity patterns between the LTPs applied might predict clinical symptoms. The intriguing question remains: How do we identify the sensitizing LTP and how do we prevent the development of (severe) food allergy as result of the induction of IgE against LTP?

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[21] - Vassilopoulou E, Zuidmeer L, Akkerdaas J, Tassios I, Rigby NR, Mills ENC, et al. Severe Immediate Allergic Reactions to Grapes: Part of a Lipid Transfer Protein-Associated Clinical Syndrome. Int Arch Allergy Immunol 2007;143:92-102

BACKGROUND: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected . OBJECTIVES: The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur . METHODS: Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens . RESULTS: All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa beta-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity . CONCLUSIONS: LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The 'LTP syndrome' is the appropriate term to describe this condition.

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[22] - Kalogeromitros DC, Makris MP, Gregoriou SG, Mousatou VG, Lyris NG, Tarassi KE, et al. Grape anaphylaxis: a study of 11 adult onset cases. Allergy Asthma Proc 2005;26:53-58

Reports of immunoglobulin E (IgE)-mediated allergic reactions to grapes and wine are limited in the literature. Nevertheless, grapes are widely grown and consumed in Mediterranean countries. The object of this prospective study was to present clinical features, in vivo and in vitro allergy testing, and human leukocyte antigen (HLA) serotyping in patients with recurring reactions to grapes and grape products. Eleven unrelated Greek patients, six men and five women (aged 16-44 years; mean, 26.9 years) were enrolled based on a documented history of IgE-mediated reactions to grapes, wine, or other grape products. Their evaluation included full history, reaction severity, clinical examination, skin-prick tests with food allergens and molds, serum IgE, specific IgEs to the same allergen battery, and HLA typing. Patients reported 35 grape-induced anaphylaxis episodes ranging from moderate (more than one system involved but not prominent respiratory or cardiovascular symptoms; 45.5%) to severe (serious respiratory obstruction and/or hypotension and loss of consciousness; 54.5%). A causative agent was identified: wine, 10/35 (28.6%); red grapes, 9/35 (25.7%); stuffed vine leaves, 8/35 (22.9%); raisins, 3/35 (8.6%); white grapes, 2/35 (5.7%); wine vinegar, 2/35 (5. 7%); and grape juice, 1/35 (2.9%). Other foods that induced anaphylaxis were apples (54.5%), cherries (18.6%), peaches (18.6%), and bananas (9.3%). Specific IgE values were in accordance with skin-prick tests reactivity. Concerning HLA typing, 9/11 possessed HLA-DR11(5) and -DQ7(3) and the remaining two possessed HLA-DR17(3) and -DQ2 antigens. Grapes, wine and other grape products might cause serious allergic reactions in sensitized individuals. The cosensitization and reaction incidence to other fruit allergens could be a basis for further investigation of panallergens of fruits. HLA class II antigens may contribute in genetic predisposition to these allergic reactions.

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[23] - Reekers R, Busche M, Wittmann M, Kapp A, Werfel T. Birch pollen-related foods trigger atopic dermatitis in patients with specific cutaneous T-cell responses to birch pollen antigens. J Allergy Clin Immunol 1999;104:466-472

BACKGROUND: Patients with inhalant allergy caused by birch pollen frequently demonstrate immediate symptoms to cross-reactive fruits, vegetables, or both. The nature of late eczematous reactions to pollen related food antigens has not been investigated in detail. OBJECTIVE: The purpose of this study was to find out whether isolated late eczematous reactions to birch pollen-related food antigens can be observed in patients with atopic dermatitis (AD) who are highly sensitized to birch pollen antigens. A possible linkage of such reactions with specific T-cell responses to birch pollen antigens in the blood and lesional skin was examined as well. METHODS: We examined 37 adult patients with AD and hypersensitivity to birch pollen but without any history of immediate responses to food challenges. These patients underwent an elimination diet, including all birch pollen-related food antigens, followed by a double-blind, placebo-controlled, oral provocation. Blood and skin biopsy specimens were taken to examine a birch pollen-specific lymphocyte response. RESULTS: Seventeen patients reacted with a deterioration of AD symptoms. Food- or birch pollen-specific IgE did not differentiate these patients from nonreactive patients. A significantly higher increase in the proportion of blood lymphocytes expressing the cutaneous lymphocyte antigen on incubation with birch pollen antigens was found in cells from reactive compared with nonreactive patients. The proliferative response of skin-derived T-cell lines from reactive patients to birch pollen extract or Bet v 1 was significantly higher than that of nonreactive patients. An enrichment of more than 25% of T-lymphocyte subpopulations defined by T-cell receptor-Vbeta elements was detected in the majority of such antigen-stimulated T-cell lines from responsive patients. A higher frequency of birch pollen-reactive T cells was calculated from limiting-dilution assays, and a higher rate of birch pollen-specific T-cell clones was generated from cultures with skin-derived T cells from reactive patients. CONCLUSION: Our results show, for the first time, that a subpopulation of patients with hypersensitivity to birch pollen and AD reacts with worsening of eczema after oral challenge with birch pollen-related foods and that a birch pollen-specific T-cell response can be found in the lesional skin of these patients.

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[24] - Breuer K, Wulf A, Constien A, Tetau D, Kapp A, Werfel T. Birch pollen-related food as a provocation factor of allergic symptoms in children with atopic eczema/dermatitis syndrome. Allergy 2004;59:988-994

BACKGROUND: Food allergy to cow's milk or hen's egg is a common problem in children with atopic eczema/dermatitis syndrome (AEDS) but the role of birch pollen-related food for the induction of allergic symptoms is still not clear. PATIENTS/METHODS: Twelve children (median age 5 years) with AEDS underwent an oral challenge with those birch pollen-related foods which were reported to induce no immediate symptoms, but were consumed on a regular basis. Total IgE and specific IgE to birch pollen, Bet v 1/2 and various birch pollen-related foods were determined . RESULTS: Seven of 12 children showed immediate and/or late eczematous reactions upon ingestion of birch pollen-related foodstuff. Four children showed a worsening of eczema 24 h upon oral challenge with a significant difference in SCORAD before and after challenge. There were no differences in terms of total IgE or birch pollen-specific IgE between children with a late eczematous response and non-reacting children . CONCLUSIONS: Birch pollen-related food may induce allergic symptoms in children with AEDS who exhibit a sensitization to birch pollen. Oral challenge tests should be performed in those children who suffer from severe AEDS and who are highly sensitized to birch pollen allergens even in the absence of a history suggestive of food allergy.

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[25] - Bohle B. The impact of pollen-related food allergens on pollen allergy. Allergy 2007;62:3-10

Patients with birch pollen allergy frequently develop hypersensitivity reactions to certain foods, e.g. apples, celery, carrots and hazelnuts. These reactions are mainly caused by IgE-antibodies specific for the major birch pollen allergen, Bet v 1, which cross-react with homologous proteins in these foods. Analyzing the T-cell response to Bet v 1-related food allergens revealed that these dietary proteins contain several distinct T-cell epitopes and activate Bet v 1-specific T cells to proliferate and produce cytokines. Several of these cross-reactive T-cell epitopes were not destroyed by simulated gastrointestinal digestion of food allergens and stimulated Bet v 1-specific T cells despite nonreactivity with IgE antibodies. Similarly, cooked food allergens did not elicit IgE-mediated symptoms (oral allergy syndromes) but caused T-cell-mediated late-phase reactions (deterioration of atopic eczema) in birch pollen-allergic patients with atopic dermatitis because thermal processing affected their conformational structure and not the primary amino acid sequence. Thus, T-cell cross-reactivity between Bet v 1 and related food allergens occurs independently of IgE-cross-reactivity in vitro and in vivo. We speculate that symptom-free consumption of pollen-related food allergens may have implications for the pollen-specific immune response of allergic individuals.

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[26] - Vieths S, Schöning B, Petersen A. Characterization of the 18-kDa apple allergen by two-dimensional immunoblotting and microsequencing. Int Arch Allergy Immunol 1994;104:399-404

A low-temperature extract taken from Golden Delicious apples was separated by two-dimensional polyacrylamide gel electrophoresis. By means of two-dimensional immunoblotting with patients' serum containing IgE specific to Bet v I, a rabbit polyclonal antiserum raised against Bet v I, and two Bet v I specific monoclonal antibodies, epitopes cross-reactive to Bet v I were identified on an apple allergen with a molecular mass of 18 kDa and pI 5.5. Furthermore, certain antibody reactivities with 4 isoproteins of a molecular mass of 16 kDa and pIs ranging from 4.9 to 5.5 were observed, which may indicate the presence of Bet v I related epitopes on these proteins. Based on 26 amino acid residues, N-terminal sequencing of the 18-kDa apple allergen revealed 62% sequence identity between Bet v I from birch pollen and the apple allergen. Our results therefore support the view that both proteins express common as well as non-related IgE-reactive epitopes.

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[27] - Fahlbusch B, Rudeschko O, Müller WD, Schlenvoigt G, Vettermann S, Jäger L. Purification and characterization of the major allergen from apple and its allergenic cross-reactivity with Bet v 1. Int Arch Allergy Immunol 1995;108:119-126

The major allergen from apple extract was concentrated by anion exchange chromatography and further purified by reverse-phase HPLC. A distinct peak with a high degree of homogeneity was obtained. The isolated protein has a MW of 18 kD and specific IgE-binding capacity (immunoblotting, IgE-binding inhibition). N-terminal amino acid analyses of the allergen allowed 37 cleavages and showed 67.6% identity to Bet v 1, the major allergen of birch pollen. Enzyme immunoassay inhibition studies with serum of birch/apple-allergic patients showed that besides cross-reacting structures to Bet v 1, apple-specific IgE antibodies could exist. Monoclonal antibodies (mAbs) were raised against the 18-kD allergen from apple and characterized by means of immunoblotting and ELISA. Only three of the eight studied mAbs reacted with Bet v 1.

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[28] - Vanek-Krebitz M, Hoffmann-Sommergruber K, Laimer da Camara Machado M, Susani M, Ebner C, Kraft D, et al. Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen allergen. Biochem Biophys Res Commun 1995;214:538-551

The number of tree-pollen-allergic patients showing allergic reactions to apples, various vegetables and nuts is increasing. In this paper the molecular characterization of the major apple allergen, Mal d 1, is reported. The cDNA coding for Mal d 1 was cloned and sequenced. Its open reading frame codes for a protein of 159 amino acids with a predicted molecular mass of 17.7 kDa and a predicted pI of 5.9. Sequence comparison to Bet v 1, the major birch pollen allergen, revealed 64.5% identity on the amino acid level and 55.6% identity on the nucleic acid level. Recombinant Mal d 1 was expressed in the plasmid pMW 175 in E. coli BL 21 (DE3) and its immunological properties were tested. Crossreactivity with Bet v 1 was shown by inhibition assays.

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[29] - Vieths S, Janek K, Aulepp H, Petersen A. Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I). Allergy 1995;50:421-430

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith applies were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients' sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Furthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.

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[30] - Ebner C, Hirschwehr R, Bauer L, Breiteneder H, Valenta R, Ebner H, et al. Identification of allergens in fruits and vegetables: IgE cross-reactivities with the important birch pollen allergens Bet v 1 and Bet v 2 (birch profilin). J Allergy Clin Immunol 1995;95:962-969

In this study serum samples collected from 20 patients with birch pollen allergy were investigated. All patients had experienced allergic symptoms after contact with or ingestion of particular fresh fruits and vegetables known as birch pollen-related foods. METHODS: Serum samples were tested by means of immunoblotting for IgE reactivities with proteins in extracts of birch pollen, apple, pear, celery, carrot, and potato. Anti-Bet v 1 and anti-Bet v 2 antibodies were used to investigate cross-reactivity. Inhibition studies were performed by preincubation of sera with recombinant Bet v 1 and Bet v 2. RESULTS: IgE binding to proteins, corresponding to the major birch pollen allergen Bet v 1 and to Bet v 2 (birch pollen profilin) could be observed. An allergen homologous to Bet v 1 could be detected in apple, pear, and celery when a Bet v 1-specific monoclonal antibody was used. Testing a polyclonal rabbit anti-Bet v 2 antibody with extracts of the respective plants revealed the presence of profilins in every source tested. Inhibition with recombinant Bet v 1 and Bet v 2 led to complete blocking or marked reduction of IgE binding to proteins of comparable molecular weights in the respective food extracts, indicating IgE cross-reactivity. CONCLUSION: Our results indicate that many plant-derived food agents contain proteins with high homology to the birch pollen allergens Bet v 1 and Bet v 2 and must therefore be considered as potentially threatening for patients with tree pollen allergy.

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[31] - Botton A, Lezzer P, Dorigoni A, Barcaccia G, Ruperti B, Ramina A. Genetic and Environmental Factors Affecting Allergen-Related Gene Expression in Apple Fruit (Malus domestica L. Borkh). J Agric Food Chem 2008;56:6707-6716

Freshly consumed apples can cause allergic reactions because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3, and Mal d 4, and their cross-reactivity with sensitizing allergens of other species. Knowledge of environmental and endogenous factors affecting the allergenic potential of apples would provide important information to apple breeders, growers, and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.

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[32] - Sancho AI, Foxall R, Browne T, Dey R, Zuidmeer L, Marzban G, et al. Effect of Postharvest Storage on the Expression of the Apple Allergen Mal d 1. J Agric Food Chem 2006;54:5917-5923

Consumption of fresh apples can cause allergy in susceptible individuals. A competitive enzyme-linked immunosorbent assay (ELISA) has been developed to determine Mal d 1 levels in apple pulp using a monoclonal antibody (BIP-1). The ELISA was able to rank ten cultivars according to their Mal d 1 content (between 3.8 and 72.5 mug/g pulp). For the first time, it has been demonstrated that growing conditions and postharvest storage, using three different treatments over a 5 month period in 2 consecutive years, increase Mal d 1 expression at a translational and transcriptional level (3.5- and 8.5-fold under controlled atmosphere storage). Expression of three major Mal d 1 isoforms was observed by real-time polymerase chain reaction over the 5 month storage period, and Mal d 1.02 was the most highly expressed isoform. In conclusion, Mal d 1 gene expression was significantly increased during modified atmosphere storage. Individuals suffering from birch pollen-apple allergy syndrome might experience fewer problems consuming freshly picked apples.

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[33] - Breiteneder H, Mills C. Structural bioinformatic approaches to understand cross-reactivity. Mol Nutr Food Res 2006;50:628-632

Cross-reactivity of allergens results from the presence of antibody-accessible conserved surface structures. These can best be studied when allergens have been structurally defined by X-ray crystallography or another structure determination method. When this is not the case, mimotope technology provides a useful alternative for elucidating antibody-binding sites on allergens. Structural bioinformatic approaches have been used to study the cross-reactivity of inhalant allergens with labile food allergens (Bet v 1 family) as well as the cross-reactivity between stable food allergens such as members of the nonspecific lipid transfer protein family. It was found that the degree of similarity of the structures correlated with the observed IgE cross-reactivities. However, IgE cross-reactivity between structurally unrelated allergens has not been demonstrated to date.

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[34] - Ma Y, Gadermaier G, Bohle B, Bolhaar S, Knulst A, Markovic-Housley Z, et al. Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1. Int Arch Allergy Immunol 2006;139:53-62

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 IgE 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.

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[35] - Puehringer HM, Zinoecker I, Marzban G, Katinger H, Laimer M. MdAP, a novel protein in apple, is associated with the major allergen Mal d 1. Gene 2003;321:173-183

Mal d 1, an 18-kDa intracellular pathogenesis-related protein (PR-10), has been known since long as the major apple allergen in Middle and Northern Europe. However, its biological function, as that of many other PR-10 proteins, is still unknown. In order to identify proteins putatively interacting with Mal d 1, an expression library of Malus domestica was screened using the yeast-two-hybrid (Y2H) system. A novel protein binding to two isoforms of Mal d 1 being used as 'bait' was isolated. The deduced amino acid sequence from the corresponding full-length cDNA of the predicted Mal d 1-Associated-Protein (MdAP) do not display any homology to known proteins, but shares 45% identity with a 'hypothetical protein' in Arabidopsis thaliana. Southern analysis of the apple genome indicated that MdAP, comprising 190 amino acids, is encoded by a single gene. The expression pattern of the 1-kb MdAP transcript resembled the expression profile of the different Mal d 1 isoforms in various apple organs, however at a much lower level. Furthermore, a huge variation in transcription levels of Mal d 1 isoforms was observed in apple tissue. For both, Mal d 1 and MdAP highest amounts of mRNAs were measured in ripe fruits and significantly lower amounts in vegetative tissue by quantitative reverse transcription-polymerase chain reaction (RT-PCR).

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[36] - Oh DH, Song KJ, Shih YU, Chung WI. Isolation of a cDNA encoding a 31-kda, PR5 thaumatin-like protein abundantly expressed in apple fruit. Biosci Biotechnol Biochem 2000;64:355-362

A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.

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[37] - Oberhuber C, Ma Y, Marsh J, Rigby N, Smole U, Radauer C, et al. Purification and characterisation of relevant natural and recombinant apple allergens. Mol Nutr Food Res 2008;52(suppl. 2):S208-S219

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purification.

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[38] - Smole U, Bublin M, Radauer C, Ebner C, Breiteneder H. Mal d 2, the Thaumatin-Like Allergen from Apple, Is Highly Resistant to Gastrointestinal Digestion and Thermal Processing. Int Arch Allergy Immunol 2008;147:289-298

BACKGROUND: The stability of food allergens to proteolysis and thermal denaturation contributes considerably to their allergenicity . METHODS: Mal d 2, an allergenic thaumatin-like protein (TLP) from apple, was isolated and purified by anion exchange chromatography. Its IgE reactivity was tested by ELISA and immunoblotting using sera from apple allergic patients. The proteolytic stability of Mal d 2 was investigated using two in vitro digestion models. Effects of thermal treatment at different pH values on the secondary structure of the protein were recorded by circular dichroism. The level and IgE reactivity of Mal d 2 present in industrially prepared foods were assessed . RESULTS: Purified Mal d 2 consisted of two isoforms. Both harbored carbohydrate moieties and bound serum IgE from apple allergic individuals. Mal d 2 showed remarkable stability to proteolysis and thermal treatments. The allergen remained intact after 2 h each of gastric and subsequent duodenal digestion retaining its full IgE-binding capacity. Mal d 2 was unfolded at neutral and acidic pH at 70 degrees C. Refolding after cooling was only observed at acidic pH. Mal d 2 detected by an anti-TLP antibody in cloudy apple juice did not bind IgE of a serum pool of apple allergic patients . CONCLUSION: Our findings suggest that Mal d 2 maintains its structure in the gastrointestinal tract, a feature essential for sensitizing the mucosal immune system and provoking allergic reactions.

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[39] - Marzban G, Puehringer H, Dey R, Brynda S, Ma Y, Martinelli A, et al. Localisation and distribution of the major allergens in apple fruits. Plant Sci 2005;169:387-394

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[40] - Barre A, Borges JP, Sabatier V, Culerrier R, Rancé F, Didier A, et al. Dosage des LTP et des protéines thaumatin-like dans la peau et la pulpe des fruits. Rev Fr Allergol Immunol Clin 2008;48:264

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[41] - Hsieh LS, Moos M, Lin Y. Characterization of apple 18 and 31 kD allergens by microsequencing and evaluation of their content during storage and ripening. J Allergy Clin Immunol 1995;96:960-970

Patients with tree pollinosis frequently report allergic reactions after ingestion of apples. The severity of apple allergy has been related to the variety of apples and their degree of maturity. To generate a serum pool that is representative of various IgE-binding patterns of apple-allergic sera, serum samples from 34 patients allergic to tree pollens were screened. Only 24 serum samples reacted to the apple extract. Pooled serum was used to identify allergens in apples. An efficient and consistent extraction method for apple fruits was used to compare the immunoreactivities of extracts of different varieties (McIntosh, Red Delicious, Granny Smith, and Golden Delicious) of freshly picked and store-purchased apples. We found that Golden Delicious apples had the greatest amount of the 18 kd allergen, which has been reported to be a potent IgE-binding apple allergen. Store-purchased apples contained higher concentrations of the 18 kd allergen than freshly picked apples. In our study only 37.5% of sera reacted to the 18 kd protein, whereas 75% of the sera reacted to a 31 kd allergen. Other immunoreactive bands in apple extracts included proteins of 50, 38, 16, 14, and 13 kd. The amino-terminal amino acid sequences of the two major allergens, 18 kd and 31 kd, were determined. These sequences shared approximately 50% identity with disease resistance proteins of various plants or Bet v 1 in birch tree pollens. The appearance of various allergens was also investigated in mature apples during storage. The amount of 18 kd allergen increased significantly when apples were stored at 4 degrees C. However, under controlled atmospheric conditions in which oxygen- and carbon dioxide-induced ripening were regulated, the amount of 18 kd allergen remained unaffected. Because ripening and maturation were not associated with increases in 18 kd allergen content, the observed changes might be induced by factors related to disease resistance.

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[42] - Zuidmeer L, Bolhaar S, Knulst A, van Leeuwen WA, Aalbers M, Krebitz M, et al. Severe symptoms caused by thaumatin-like proteins in foods: a result of primary sensitization to birch pollen ? EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°196

Background: It has almost become a dogma that birch pollen-related food allergies are always mild, because Bet v 1 and its homologues from foods as well as profilins are extremely heat and protease-sensitive. Protease-resistant birch pollen allergens have so far not been reported. Methods: A panel of 477 sera from The Netherlands with >2.0 IU/ml of specific IgE against apple were analysed by RAST for specific IgE against 4 apple allergens: nMal d 1, rMal d 2 (thaumatin-like protein [TLP]), nMal d 3 (LTP) and nMal d 4 (profilin). rMal d 2-positive sera (>0.7 IU/ml) were further analysed by RAST inhibition with (protease-digested) birch pollen extract. Serum from a patient with severe food-related anaphylaxis was studied in more detail by RAST(-inhibition) and immunoblot(- inhibition). Results: As expected most sera contained significant titers of specific IgE against Mal d 1 (70% with > 1.0 IU/ml). Frequency of recognition of Mal d 2, 3 and 4 was much lower (23%, 9% and 19% respectively). For 55 sera with a clear positive RAST for rMal d 2 IgE-binding to this allergen was inhibited with birch pollen extract. On average the Mald 2 RAST was inhibited by 75%, indicating that birch pollen extract contains a crossreactive structure with food TLP. A similar inhibition was observed with protease-digested birch pollen extract, suggesting that the putative TLP-related allergen from birch pollen is extremely protease resistant. This observation fits with the high degree of stability observed for rMal d 2 in simulated gastric fluid. Considering this stability, it is not surprising that among the patients with TLP-specific IgE antibodies, severe food allergic symptoms were observed. One of these patients was further analysed in depth. This patient experienced anaphylactic shock upon consumption of hazelnut liquor, raspberry and several other fruits on different occasions. IgE binding to raspberry and several other foods could be inhibited by birch pollen and by rMal d 2 in both RAST- and immunoblot-inhibition, indicating that the severe food allergy of this patient is caused by TLP-like allergens and that sensitisation is most likely birch pollen driven. Conclusion: Birch pollen contain an allergen that is crossreactive with food TLPs and is most likely responsible for sensitisation. In contrast to most Bet v 1-related allergens, food TLPs can cause severe food allergy.

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[43] - Smole U, Bublin M, Radauer C, Ebner C, Scheiner O, Breiteneder H. Allergenic Fruit TLPs Possess Different Degrees of IgE Crossreactivity. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°195

RATIONALE: Thaumatin-like proteins (TLPs) are important allergens of a number of plant foods. We aimed to examine the IgE cross-reactivity between the apple TLP Mal d 2 and TLPs from kiwi (Act c2), grape (VvTLP), and cherry (Pru av 2) METHODS: Amino acid sequences of apple, cherry, kiwi, and grape TLPs were aligned. The TLPs were purified. IgE ELISA and ELISA inhibitions were carried out using sera from 46 apple allergic patients. The relevance of N-glycosylation for IgE-binding was investigated by inhibition with the oligosaccharide BSA-MUXF RESULTS: Sequence alignments showed 33% identity of Mal d 2 to Act c 2, 35% to VvTLP, and 69 % to Pru av 2. Mal d 2 was IgE reactive with a prevalence of 39% (18/46). Nine of these 18 patients recognized N-glycan epitopes on Mal d 2, the 9 remaining showed binding to protein epitopes Two of the 9 protein-specific sera had IgE to all four TLPs, 6/9 to Mal d 2, Act c 2, and Pru av 2, and one to Mal d 2 and Pru av 2. In addition, Mal d 2 and Pru av 2 showed a high extent of cross-reactivity CONCLUSIONS: The glycoallergen Mal d 2 shares cross-reactive epitopes with other TLPs that encompass the Mal d 2 polypeptide and its N-glycan structure. Inhibition of IgE-Mal d 2 interactions with Pru av 2 demonstrated that many of the epitopes of Mal d 2 were cross-reactive with epitopes on Pru av 2 This work was supported by the Austrian Science Fund grant SFBF01802 Funding: Austrian Science Fund grant SFB-F01802

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[44] - Smole U, Bublin M, Radauer C, Ebner C, Breiteneder H. Mal d 2, the Thaumatin-Like Allergen from Apple, Is Highly Resistant to Gastrointestinal Digestion and Thermal Processing. Int Arch Allergy Immunol 2008;147:289-298

BACKGROUND: The stability of food allergens to proteolysis and thermal denaturation contributes considerably to their allergenicity . METHODS: Mal d 2, an allergenic thaumatin-like protein (TLP) from apple, was isolated and purified by anion exchange chromatography. Its IgE reactivity was tested by ELISA and immunoblotting using sera from apple allergic patients. The proteolytic stability of Mal d 2 was investigated using two in vitro digestion models. Effects of thermal treatment at different pH values on the secondary structure of the protein were recorded by circular dichroism. The level and IgE reactivity of Mal d 2 present in industrially prepared foods were assessed . RESULTS: Purified Mal d 2 consisted of two isoforms. Both harbored carbohydrate moieties and bound serum IgE from apple allergic individuals. Mal d 2 showed remarkable stability to proteolysis and thermal treatments. The allergen remained intact after 2 h each of gastric and subsequent duodenal digestion retaining its full IgE-binding capacity. Mal d 2 was unfolded at neutral and acidic pH at 70 degrees C. Refolding after cooling was only observed at acidic pH. Mal d 2 detected by an anti-TLP antibody in cloudy apple juice did not bind IgE of a serum pool of apple allergic patients . CONCLUSION: Our findings suggest that Mal d 2 maintains its structure in the gastrointestinal tract, a feature essential for sensitizing the mucosal immune system and provoking allergic reactions.

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[45] - Borges JP, Jauneau A, Brule C, Culerrier R, Barre A, Didier A, et al. The lipid transfer proteins (LTP) essentially concentrate in the skin of Rosaceae fruits as cell surface exposed allergens. Plant Physiol Biochem 2006;44:535-542

The localization and distribution of non-specific lipid transfer proteins (nsLTP) allergens in the skin and pulp of Rosaceae fruits (apple, peach, apricot, plum) has been investigated. nsLTP essentially concentrate in the pericarp of the fruits whereas the pulp contains lower amounts of allergens. Immunolocalization showed they are primarily located in the cytosol but are subsequently excreted and finally accumulate at the plasmalemma-cell wall interface and in the cell wall. However, high discrepancies were observed in the content of allergens among, e.g. different cultivars of apple. As a consequence, the consumption of peeled-off fruits is recommended to reduce the risk of severe allergic reactions (anaphylactic shock) in individuals sensitized to Rosaceae fruits.

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[46] - Fernández-Rivas M, Gonzalez-Mancebo E, Van Leeuwen A, Zuidmeer L, Rigby N, Ma Y, et al. Apple lipid transfer protein (Mal d 3) and profilin (Mal d 4) are the clinically relevant allergens in Spanish patients allergic to apple. Allergy Clin Immunol Int 2005;17(Suppl. 1):339

Research, Physical Biochemistry unit, Norwich, United Kingdom; 4University of Vienna, Pathophysiology department, Vienna, Austria Background. Four apple allergens have been identified so far: Mal d 1 ( Bet v 1 homologue), Mal d 2 (thaumatin-like protein), Mal d 3 (non-specific lipid transfer protein), and Mal d 4 (profilin). Mal d 1 is the major allergen in patients with apple allergy linked to birch pollinosis, and Mal d 3 is the major allergen in patients from Spain and Italy without birch pollinosis. Aim. To investigate the clinical relevance of the 4 apple allergens in allergic patients from Spain. Methods. Medical history, skin prick tests (SPTs) with fresh apple, and RAST to apple, nMal d 1, rMal d 2, nMal d 3, and rMal d 4, were carried out in patients reporting adverse reactions to apple. Clinical reactivity was confirmed by a double-blind placebo-controlled apple challenge. Results. Apple allergy was confirmed in 77 patients, 49 females, 28 males, mean age 23.3 years. Pollen allergy was present in 86%. Isolated oropharyngeal symptoms (OAS) were reported by 61% of the patients, whereas 39% had systemic symptoms (urticaria, anaphylaxis). All subjects presented positive SPTs to apple. Mean RAST values to apple, nMal d 1, rMal d 2, nMal d 3, and rMal d 4 were 5.17, 0.63, 0.78, 3.46, and 2.56 IU/ml, respectively. Positive (“0.8 IU/ ml) RAST results to nMal d 1, rMal d 2, nMal d 3, and rMal d 4 were found in 14.5%, 25%, 59.2% and 40.8% of patients, respectively. With the combination of Mal d 3 and 4, 86.8% of allergic patients were detected; with the further addition of Mal d 2, 89.5% of allergic subjects were identified. The inclusion of Mal d 1 did not increase this figure. A significant association was found between systemic symptoms induced by apple and sensitisation to Mal d 3, with an OR of 2.95 (1.13-7.67), and between OAS and sensitisation to rMal d 4, with an OR of 5.34 (1.94-14.62). Furthermore, 26 patients were monosensitised to nMal d 3, and 12 subjects to rMald 4. In this latter group isolated mild OAS was observed in 92% of patients, whereas it was only present in 31% of the subjects monosensitised to nMal d 3 (p<0.001). No associations were found between the sensitisation to Mal d 1 and 2 and the clinical presentation of apple allergy. Conclusions. Mal d 3 and Mal d 4 are the relevant allergens in Spanish patients allergic to apple. Mal d 3 is related to severe systemic reactions, whereas Mal d 4 is involved in mild local OAS. Mal d 1 and 2 do not seem to be clinically relevant apple allergens in Spanish patients.

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[47] - Asero R, Mistrello G, Roncarolo D, Amato S, Zanoni D, Barocci F, et al. Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. J Allergy Clin Immunol 2003;112:427-432

BACKGROUND: A proper classification of patients allergic to plant-derived foods is of pivotal importance because the clinical features of allergic reactions to fruits and vegetables depend on the nature and characteristics of proteins responsible for sensitization. However, in normal clinical settings this is presently impossible . OBJECTIVE: We sought to detect clinical markers of sensitization to profilin . METHODS: Seventy-one patients allergic to fruits and vegetables but not sensitized to lipid transfer protein or natural rubber latex were studied. Food allergy was ascertained on the basis of clinical history and positive skin prick test responses with fresh foods, commercial extracts, or both. Allergies to foods that had caused less than 2 adverse reactions were confirmed by means of open oral challenge. IgE reactivity to rBet v 1/rBet v 2 and to natural Phleum species profilin were detected. Moreover, IgE to the 30- to 40-kd and 60- to 90-kd birch pollen-enriched fractions, which also can be involved in cross-reactivity phenomena, were measured in sera from 52 patients by means of ELISA . RESULTS: On the basis of in vitro tests, 24, 18, and 25 patients turned out to be sensitized to Bet v 1, Bet v 2, or both, respectively. Four patients had negative test results for both allergens. Hypersensitivity to Bet v 2 was strongly associated with clinical allergy to citrus fruits (39% in patients monosensitized to Bet v 2 vs 4% in patients monosensitized to Bet v 1, P <.025), melon or watermelon (67% vs 0%, P <.001), banana (66% vs 8%, P <.001), and tomato (33% vs 0%, P <.05), whereas Bet v 1 sensitivity was associated with clinical allergy to apple (100% vs 39%, P <.001) and hazelnut (56% vs 0%, P <.001). The sensitivity of a history of allergy to gourd fruits, citrus fruits, tomato, banana, or a combination thereof as a means to detect profilin-hypersensitive patients was 85% (41/48). The specificity of an allergy to any of these fruits exceeded 85%, with positive predictive values ranging between 68% and 91% . CONCLUSION: In clinical settings in which laboratory investigations are not easily accessible, allergy to melon, watermelon, citrus fruits, tomato, and banana can be used as a marker of profilin hypersensitivity once a sensitization to natural rubber latex and lipid transfer protein is ruled out.

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[48] - Karamloo F, Wangorsch A, Kasahara H, Davin LB, Haustein D, Lewis NG, et al. Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables. Eur J Biochem 2001;268:5310-5320

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.

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[49] - Bolhaar STHP, Zuidmeer L, Ma Y, Ferreira F, Bruijnzeel-Koomen CAFM, Hoffmann-Sommergruber K, et al. A mutant of the major apple allergen, Mal d 1, demonstrating hypo-allergenicity in the target organ by double-blind placebo-controlled food challenge. Clin Exp Allergy 2005;35:1638-1644

BACKGROUND: Allergen-specific immunotherapy for food allergy has been hindered by severe side-effects in the past. Well-characterized hypo-allergenic recombinant food allergens potentially offer a safe solution . OBJECTIVE: To demonstrate hypo-allergenicity of a mutated major food allergen from apple, Mal d 1, in vitro and in vivo . METHODS: A mutant of the major apple allergen, Mal d 1, was obtained by site-directed mutagenesis exchanging five amino acid residues. Fourteen patients with combined birch pollen-related apple allergy were included in the study. Hypo-allergenicity of the mutant rMal d 1 (rMal d 1mut) compared with rMal d 1 was assessed by in vitro methods, i.e. RAST (inhibition), immunoblotting and basophil histamine release (BHR) and in vivo by skin prick test and double-blind placebo-controlled food challenge (DBPCFC) . RESULTS: RAST analysis (n = 14) revealed that IgE reactivity to rMal d 1mut was twofold lower than that of the wild-type molecule (95% confidence interval (CI): 1.7-2.4). RAST inhibition (n = 6) showed a 7.8-fold decrease in IgE-binding potency (95% CI: 3.0-12.6). In contrast to this moderate decrease in IgE-binding potency, the biological activity of rMal d 1mut assessed by SPT and BHR decreased 10-200-fold. Hypo-allergenicity was confirmed by DBPCFC (n = 2) with both recombinant molecules . CONCLUSION: A moderate decrease in IgE-binding potency translates into a potent inhibition of biological activity. This is the first study that confirms by DBPCFC that a mutated recombinant major food allergen is clinically hypo-allergenic. This paves the way towards safer immunotherapy for the treatment of food-allergic patients.

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[50] - Ma Y, Gadermaier G, Bohle B, Bolhaar S, Knulst A, Markovic-Housley Z, et al. Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1. Int Arch Allergy Immunol 2006;139:53-62

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 IgE 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.

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[51] - Gilissen LJWJ, Bolhaar STHP, Matos CI, Rouwendal GJA, Boone MJ, Krens FA, et al. Silencing the major apple allergen Mal d 1 by using the RNA interference approach. J Allergy Clin Immunol 2005;115:364-369

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions . OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference . METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1 . RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made . CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.

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[52] - Marzban G, Puehringer H, Dey R, Brynda S, Ma Y, Martinelli A, et al. Localisation and distribution of the major allergens in apple fruits. Plant Sci 2005;169:387-394

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[53] - Asero R, Marzban G, Martinelli A, Zaccarini M, Machado ML. Search for low allergenic apple cultivars for birch pollen-allergic patients: is there a correlation between in vitro assays and patient response ? Eur Ann Allergy Clin Immunol 2006;38:94-98

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.

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[54] - Botton A, Lezzer P, Dorigoni A, Barcaccia G, Ruperti B, Ramina A. Genetic and Environmental Factors Affecting Allergen-Related Gene Expression in Apple Fruit (Malus domestica L. Borkh). J Agric Food Chem 2008;56:6707-6716

Freshly consumed apples can cause allergic reactions because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3, and Mal d 4, and their cross-reactivity with sensitizing allergens of other species. Knowledge of environmental and endogenous factors affecting the allergenic potential of apples would provide important information to apple breeders, growers, and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.

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[55] - Sancho AI, van Ree R, van Leeuwen A, Meulenbroek BJ, van de Weg EW, Gilissen LJWJ, et al. Measurement of Lipid Transfer Protein in 88 Apple Cultivars. Int Arch Allergy Immunol 2008;146:19-26

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars . OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars . METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC) . RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels . CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

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[56] - Goodman RE. Practical and predictive bioinformatics methods for the identification of potentially cross-reactive protein matches. Mol Nutr Food Res 2006;50:655-660

A bioinformatics comparison of proteins introduced into food crops through genetic engineering provides a mechanism to identify those proteins that may present an increased risk of allergic reactions for individuals with existing allergies. The goal is to identify proteins that are known to be allergens or are so similar to an allergen that they may induce allergic cross-reactions. Three comparative approaches have traditionally been used, or considered for safety evaluations. One identifies any short (6-8) amino acid segment of the protein that exactly matches a known allergen sequence. The second is an overall primary sequence comparison using Basic Local Alignment Search Tool (BLAST) or FASTA to find matches of greater than 35% identity over 80 amino acids. The third is based on 3-D prediction programs to identify 3-D similarities that might predict potential cross-reactivity. The utility of each of these approaches was debated in the bioinformatics workshop. The consensus agreement from the expert workshop participants was that the short-segment match (e. g., 6-8 amino acids) provides an unacceptably high rate of false positive matches and an uncertain rate of true positive matches, and was not particularly useful for an allergenicity evaluation performed in the context of comprehensive safety evaluation. There was no consensus regarding the most appropriate bioinformatics method, an acceptable scoring criteria for triggering closer examination subsequent to a positive match, or an acceptable scoring mechanism for ranking the utility of the various 3-D approaches that were discussed during the workshop. However, the general consensus was that the most practical approach at this time is to evaluate primary sequence identities to known allergens using either FASTA or BLAST. While there was good agreement that identities of greater than 35% over 80 or more amino acids (recommended by Codex in 2003) is quite conservative, the conclusion was that additional data or studies would be needed to justify changing this criterion as there is some evidence that some individuals sensitized to proteins in evolutionarily conserved protein families may experience cross-reactions to proteins sharing approximately 40% identity.

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[57] - Marzban G, Puehringer H, Dey R, Brynda S, Ma Y, Martinelli A, et al. Localisation and distribution of the major allergens in apple fruits. Plant Sci 2005;169:387-394

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[58] - Zuidmeer L, van Leeuwen WA, Kleine Budde I, Breiteneder H, Ma Y, Mills C, et al. Allergenicity Assessment of Apple Cultivars: Hurdles in Quantifying Labile Fruit Allergens. Int Arch Allergy Immunol 2006;141:230-240

BACKGROUND: Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes . METHODS: Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS) . RESULTS: Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by approximately 1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen . CONCLUSIONS: Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits.

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[59] - Marzban G, Herndl A, Maghuly F, Katinger H, Laimer M. Mapping of fruit allergens by 2D electrophoresis and immunodetection. Expert Rev Proteomics 2008;5:61-75

Proteomic analyses of fruits are confronted with a series of specific obstacles: a general low protein content in plant tissues, allergen extraction from highly complex matrices and protein determination in the presence of interfering compounds. Different methods are currently being introduced to achieve higher protein yields and a simultaneous removal of interfering substances, such as polyphenols and polysaccharides. However, no universal protocol suitable for protein purification from any given plant species is available. Protein profiling by 2DE-western blotting offers a powerful tool for the detection and characterization of known and novel plant allergens. Moreover, the detection of IgE-reactive proteins from fruits is improved by combining western blot and alternative visualization techniques. The recent developments in bioinformatics and databases facilitate the interpretation of profiling studies with regard to novel potential fruit allergens

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[60] - Asero R, Marzban G, Martinelli A, Zaccarini M, Machado ML. Search for low allergenic apple cultivars for birch pollen-allergic patients: is there a correlation between in vitro assays and patient response ? Eur Ann Allergy Clin Immunol 2006;38:94-98

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.

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[61] - Sancho AI, Foxall R, Browne T, Dey R, Zuidmeer L, Marzban G, et al. Effect of Postharvest Storage on the Expression of the Apple Allergen Mal d 1. J Agric Food Chem 2006;54:5917-5923

Consumption of fresh apples can cause allergy in susceptible individuals. A competitive enzyme-linked immunosorbent assay (ELISA) has been developed to determine Mal d 1 levels in apple pulp using a monoclonal antibody (BIP-1). The ELISA was able to rank ten cultivars according to their Mal d 1 content (between 3.8 and 72.5 mug/g pulp). For the first time, it has been demonstrated that growing conditions and postharvest storage, using three different treatments over a 5 month period in 2 consecutive years, increase Mal d 1 expression at a translational and transcriptional level (3.5- and 8.5-fold under controlled atmosphere storage). Expression of three major Mal d 1 isoforms was observed by real-time polymerase chain reaction over the 5 month storage period, and Mal d 1.02 was the most highly expressed isoform. In conclusion, Mal d 1 gene expression was significantly increased during modified atmosphere storage. Individuals suffering from birch pollen-apple allergy syndrome might experience fewer problems consuming freshly picked apples.

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[62] - Marzban G, Puehringer H, Dey R, Brynda S, Ma Y, Martinelli A, et al. Localisation and distribution of the major allergens in apple fruits. Plant Sci 2005;169:387-394

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[63] - Carnes J, Ferrer A, Fernandez-Caldas E. Allergenicity of 10 different apple varieties. Ann Allergy Asthma Immunol 2006;96:564-570

BACKGROUND: More than 100 apple varieties are consumed worldwide. However, the allergenic composition of most apple varieties has not been fully characterized. OBJECTIVE: To analyze the antigenic and allergenic profiles of 10 different commercially available apple varieties. METHODS: Golden, Golden Perlim, Reineta, Reineta Parda, Fuji, Verdedoncella, Granny-Smith, Pink Lady, Royal Gala, and Starking apple varieties were carefully peeled. Peels were individually extracted and centrifuged, and the supernatants were collected, dialyzed, filtered, frozen, and freeze-dried. The extracts were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein content was measured using the Lowry-Biuret method. Twenty-two Spanish patients with oral allergy syndrome after apple ingestion were included in the study. Specific IgE to all apple varieties and birch pollen was evaluated. Eleven patients underwent skin prick testing using extracts of Golden, Starking, and Reineta Parda varieties to determine differences in their in vivo allergenicity. RESULTS: The antigenic profile of the 10 varieties showed differences using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 9-kDa band, which could correspond to Mal d 3, was visualized in all the extracts. The Starking variety had the highest protein content. Higher specific IgE values were also obtained for the Starking variety, and the lowest values were for Reineta Parda and Royal Gala. The largest wheals were obtained in all patients with the Starking variety, and the smallest with Reineta Parda. Fourteen patients (64%) recognized Mal d 3 in the Golden variety using immunoblots; 6 patients (27%), who also had specific IgE to Betula, recognized bands with higher molecular weights. CONCLUSIONS: We demonstrated differences in the antigenic and allergenic profiles of the analyzed varieties and a significant variation in Mal d 3 content.

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[64] - Borges JP, Jauneau A, Brule C, Culerrier R, Barre A, Didier A, et al. The lipid transfer proteins (LTP) essentially concentrate in the skin of Rosaceae fruits as cell surface exposed allergens. Plant Physiol Biochem 2006;44:535-542

The localization and distribution of non-specific lipid transfer proteins (nsLTP) allergens in the skin and pulp of Rosaceae fruits (apple, peach, apricot, plum) has been investigated. nsLTP essentially concentrate in the pericarp of the fruits whereas the pulp contains lower amounts of allergens. Immunolocalization showed they are primarily located in the cytosol but are subsequently excreted and finally accumulate at the plasmalemma-cell wall interface and in the cell wall. However, high discrepancies were observed in the content of allergens among, e.g. different cultivars of apple. As a consequence, the consumption of peeled-off fruits is recommended to reduce the risk of severe allergic reactions (anaphylactic shock) in individuals sensitized to Rosaceae fruits.

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[65] - Sancho AI, van Ree R, van Leeuwen A, Meulenbroek BJ, van de Weg EW, Gilissen LJWJ, et al. Measurement of Lipid Transfer Protein in 88 Apple Cultivars. Int Arch Allergy Immunol 2008;146:19-26

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars . OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars . METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC) . RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels . CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

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[66] - Botton A, Lezzer P, Dorigoni A, Barcaccia G, Ruperti B, Ramina A. Genetic and Environmental Factors Affecting Allergen-Related Gene Expression in Apple Fruit (Malus domestica L. Borkh). J Agric Food Chem 2008;56:6707-6716

Freshly consumed apples can cause allergic reactions because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3, and Mal d 4, and their cross-reactivity with sensitizing allergens of other species. Knowledge of environmental and endogenous factors affecting the allergenic potential of apples would provide important information to apple breeders, growers, and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.

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[67] - Bolhaar STHP, van de Weg WE, van Ree R, Gonzalez-Mancebo E, Zuidmeer L, Bruijnzeel-Koomen CAFM, et al. In vivo assessment with prick-to-prick testing and double-blind, placebo-controlled food challenge of allergenicity of apple cultivars. J Allergy Clin Immunol 2005;116:1080-1086

BACKGROUND: Apple cultivars have been reported to differ in allergenicity on the basis of in vitro and skin prick tests with apple extracts . OBJECTIVES: We sought to evaluate the efficacy of the prick-to-prick method in assessing differences in allergenicity of apple cultivars and to confirm differences by means of double-blind, placebo-controlled food challenge (DBPCFC) . METHODS: Intra-assay and intracultivar variation of prick-to-prick test results were determined in 6 Dutch and 8 Spanish patients with apple allergy by using 5 apples of the cultivars Golden Delicious, Fuji, and Ecolette in duplicate. In addition, 21 cultivars were screened for allergenicity in 15 Dutch patients with birch pollen and apple allergy. Two selected cultivars (Golden Delicious and Santana) were tested with DBPCFCs. The influence of storage conditions on allergenicity was assessed in 5 cultivars . RESULTS: Intra-assay variation of skin prick testing was 3.9%, and intracultivar variation was 4.1%. A ranking of 21 cultivars was made on the basis of prick-to-prick tests in 9 patients. Apple cultivars were classified as of low, intermediate, and high allergenicity, with a significant difference between low and high allergenicity (P < .001). A significant difference in allergenicity determined between Golden Delicious and Santana cultivars (P < .05) was confirmed by means of DBPCFC. With 5 cultivars, controlled atmosphere (2.5% oxygen/1% carbon dioxide) was shown to reduce allergenicity (P < .001) by 15% compared with storage at 2 degrees C . CONCLUSIONS: Prick-to-prick testing with fresh apples is a reproducible method of assessing allergenicity. Apples can be classified as of low or high allergenicity for the majority of patients. This was confirmed by using DBPCFCs. Selection of cultivars and control of storage conditions are both viable strategies for reduction of symptoms in patients with apple allergy.

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[68] - de Groot H, de Jong NW, Vuijk MH, Gerth van Wijk R. Birch pollinosis and atopy caused by apple, peach, and hazelnut: comparison of three extraction procedures with two apple strains. Allergy 1996;51:712-718

This study aimed, first, to study the prevalence in The Netherlands of atopy caused by apple, peach, and hazelnut in patients with tree pollinosis, and, second, to compare three extraction procedures for skin prick testing with two different apple strains. Skin prick tests and RAST were performed on 79 consecutive patients with tree pollinosis, visiting the department of allergology during spring 1995. In skin prick tests, we used three different extracts (juice, freeze-dried extract, and low-temperature acetone powder extract) of two apple strains, Golden Delicious and Granny Smith. Case histories for apple, peach, and hazelnut were positive in 35 (44.3%), 23 (29%), and 35 (44.3%) patients, respectively. More than two-thirds of the patients had symptoms characteristic of oral allergy syndrome. Skin prick tests for apple, peach, and hazelnut were positive in 51 (64.6%), 61 (77.2%), and 71 (89.9%) patients, respectively. Granny Smith showed more positive skin reactions and a better agreement with clinical history than Golden Delicious, and juice was superior to the two other extraction procedures for both apple strains. RAST for apple, peach, and hazelnut was positive in 53 (68.8%), 13 (16.9%), and 31 (40.3%) patients, respectively. Concordance between skin prick test and case history was found in 77%, 52%, and 54%, for apple, peach, and hazelnut, respectively. We found a high percentage of concurrence of clinical allergy to birch pollen and apple, peach, and hazelnut, confirmed by both skin prick testing and RAST. Approximately half of these patients had symptoms (especially oral allergy syndrome) after eating these products. We also found an easy extraction procedure (juice extract) suitable for apple skin prick testing, superior even to freeze-dried extraction or the low-temperature acetone powder technique.

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[69] - Bolhaar STHP, van de Weg WE, van Ree R, Gonzalez-Mancebo E, Zuidmeer L, Bruijnzeel-Koomen CAFM, et al. In vivo assessment with prick-to-prick testing and double-blind, placebo-controlled food challenge of allergenicity of apple cultivars. J Allergy Clin Immunol 2005;116:1080-1086

BACKGROUND: Apple cultivars have been reported to differ in allergenicity on the basis of in vitro and skin prick tests with apple extracts . OBJECTIVES: We sought to evaluate the efficacy of the prick-to-prick method in assessing differences in allergenicity of apple cultivars and to confirm differences by means of double-blind, placebo-controlled food challenge (DBPCFC) . METHODS: Intra-assay and intracultivar variation of prick-to-prick test results were determined in 6 Dutch and 8 Spanish patients with apple allergy by using 5 apples of the cultivars Golden Delicious, Fuji, and Ecolette in duplicate. In addition, 21 cultivars were screened for allergenicity in 15 Dutch patients with birch pollen and apple allergy. Two selected cultivars (Golden Delicious and Santana) were tested with DBPCFCs. The influence of storage conditions on allergenicity was assessed in 5 cultivars . RESULTS: Intra-assay variation of skin prick testing was 3.9%, and intracultivar variation was 4.1%. A ranking of 21 cultivars was made on the basis of prick-to-prick tests in 9 patients. Apple cultivars were classified as of low, intermediate, and high allergenicity, with a significant difference between low and high allergenicity (P < .001). A significant difference in allergenicity determined between Golden Delicious and Santana cultivars (P < .05) was confirmed by means of DBPCFC. With 5 cultivars, controlled atmosphere (2.5% oxygen/1% carbon dioxide) was shown to reduce allergenicity (P < .001) by 15% compared with storage at 2 degrees C . CONCLUSIONS: Prick-to-prick testing with fresh apples is a reproducible method of assessing allergenicity. Apples can be classified as of low or high allergenicity for the majority of patients. This was confirmed by using DBPCFCs. Selection of cultivars and control of storage conditions are both viable strategies for reduction of symptoms in patients with apple allergy.

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[70] - Kootstra HS, Vlieg-Boerstra BJ, Dubois AE. Assessment of the reduced allergenic properties of the Santana apple. Ann Allergy Asthma Immunol 2007;99:522-525

BACKGROUND: Approximately 2% of the Northern and Central European population is allergic to apples. Because of symptoms of oral allergy syndrome, many individuals avoid eating fresh apples. A strategy to allow apple allergic individuals to eat apples is the development of hypoallergenic apple varieties. OBJECTIVE: To investigate the allergenicity of a putatively hypoallergenic apple cultivar, the Santana apple, on apple allergic individuals using a method of challenge in which loss of allergenicity is minimized. METHODS: The study population consisted of 15 apple allergic individuals, who underwent an open oral challenge with 3 different apple cultivars: Santana, Golden Delicious, and Topaz. Food challenges were performed between February 1 and May 31, 2005, during the birch tree pollen season. Sensory perception during challenges was minimized by use of a blindfold and nose clip and randomization of challenge order. Subjective symptoms were scored by a visual analog scale. RESULTS: After challenge with the Santana apple, 8 of 15 study participants (53%) developed no symptoms, which was a significantly greater proportion than after challenge with the Topaz apple (1 participant) and Golden Delicious apple (1 participant) (P = .002). The visual analog scale scores for the Santana apple (dose 1) were significantly lower than that for the Golden Delicious and Topaz apples. A total of 73% of the participants stated they would eat Santana apples in the future. CONCLUSION: The Santana apple caused significantly fewer allergic symptoms in apple allergic individuals than the Golden Delicious and Topaz apples. The Santana apple may be a good choice for most apple allergic individuals who want to eat apples.

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[71] - Asero R, Marzban G, Martinelli A, Zaccarini M, Machado ML. Search for low allergenic apple cultivars for birch pollen-allergic patients: is there a correlation between in vitro assays and patient response ? Eur Ann Allergy Clin Immunol 2006;38:94-98

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.

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[72] - Carnes J, Ferrer A, Fernandez-Caldas E. Allergenicity of 10 different apple varieties. Ann Allergy Asthma Immunol 2006;96:564-570

BACKGROUND: More than 100 apple varieties are consumed worldwide. However, the allergenic composition of most apple varieties has not been fully characterized. OBJECTIVE: To analyze the antigenic and allergenic profiles of 10 different commercially available apple varieties. METHODS: Golden, Golden Perlim, Reineta, Reineta Parda, Fuji, Verdedoncella, Granny-Smith, Pink Lady, Royal Gala, and Starking apple varieties were carefully peeled. Peels were individually extracted and centrifuged, and the supernatants were collected, dialyzed, filtered, frozen, and freeze-dried. The extracts were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein content was measured using the Lowry-Biuret method. Twenty-two Spanish patients with oral allergy syndrome after apple ingestion were included in the study. Specific IgE to all apple varieties and birch pollen was evaluated. Eleven patients underwent skin prick testing using extracts of Golden, Starking, and Reineta Parda varieties to determine differences in their in vivo allergenicity. RESULTS: The antigenic profile of the 10 varieties showed differences using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 9-kDa band, which could correspond to Mal d 3, was visualized in all the extracts. The Starking variety had the highest protein content. Higher specific IgE values were also obtained for the Starking variety, and the lowest values were for Reineta Parda and Royal Gala. The largest wheals were obtained in all patients with the Starking variety, and the smallest with Reineta Parda. Fourteen patients (64%) recognized Mal d 3 in the Golden variety using immunoblots; 6 patients (27%), who also had specific IgE to Betula, recognized bands with higher molecular weights. CONCLUSIONS: We demonstrated differences in the antigenic and allergenic profiles of the analyzed varieties and a significant variation in Mal d 3 content.

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[73] - Sancho AI, van Ree R, van Leeuwen A, Meulenbroek BJ, van de Weg EW, Gilissen LJWJ, et al. Measurement of Lipid Transfer Protein in 88 Apple Cultivars. Int Arch Allergy Immunol 2008;146:19-26

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars . OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars . METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC) . RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels . CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

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[74] - Sancho AI, van Ree R, van Leeuwen A, Meulenbroek BJ, van de Weg EW, Gilissen LJWJ, et al. Measurement of Lipid Transfer Protein in 88 Apple Cultivars. Int Arch Allergy Immunol 2008;146:19-26

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars . OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars . METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC) . RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels . CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

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[75] - Hsieh LS, Moos M, Lin Y. Characterization of apple 18 and 31 kD allergens by microsequencing and evaluation of their content during storage and ripening. J Allergy Clin Immunol 1995;96:960-970

Patients with tree pollinosis frequently report allergic reactions after ingestion of apples. The severity of apple allergy has been related to the variety of apples and their degree of maturity. To generate a serum pool that is representative of various IgE-binding patterns of apple-allergic sera, serum samples from 34 patients allergic to tree pollens were screened. Only 24 serum samples reacted to the apple extract. Pooled serum was used to identify allergens in apples. An efficient and consistent extraction method for apple fruits was used to compare the immunoreactivities of extracts of different varieties (McIntosh, Red Delicious, Granny Smith, and Golden Delicious) of freshly picked and store-purchased apples. We found that Golden Delicious apples had the greatest amount of the 18 kd allergen, which has been reported to be a potent IgE-binding apple allergen. Store-purchased apples contained higher concentrations of the 18 kd allergen than freshly picked apples. In our study only 37.5% of sera reacted to the 18 kd protein, whereas 75% of the sera reacted to a 31 kd allergen. Other immunoreactive bands in apple extracts included proteins of 50, 38, 16, 14, and 13 kd. The amino-terminal amino acid sequences of the two major allergens, 18 kd and 31 kd, were determined. These sequences shared approximately 50% identity with disease resistance proteins of various plants or Bet v 1 in birch tree pollens. The appearance of various allergens was also investigated in mature apples during storage. The amount of 18 kd allergen increased significantly when apples were stored at 4 degrees C. However, under controlled atmospheric conditions in which oxygen- and carbon dioxide-induced ripening were regulated, the amount of 18 kd allergen remained unaffected. Because ripening and maturation were not associated with increases in 18 kd allergen content, the observed changes might be induced by factors related to disease resistance.

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[76] - Sancho AI, Foxall R, Browne T, Dey R, Zuidmeer L, Marzban G, et al. Effect of Postharvest Storage on the Expression of the Apple Allergen Mal d 1. J Agric Food Chem 2006;54:5917-5923

Consumption of fresh apples can cause allergy in susceptible individuals. A competitive enzyme-linked immunosorbent assay (ELISA) has been developed to determine Mal d 1 levels in apple pulp using a monoclonal antibody (BIP-1). The ELISA was able to rank ten cultivars according to their Mal d 1 content (between 3.8 and 72.5 mug/g pulp). For the first time, it has been demonstrated that growing conditions and postharvest storage, using three different treatments over a 5 month period in 2 consecutive years, increase Mal d 1 expression at a translational and transcriptional level (3.5- and 8.5-fold under controlled atmosphere storage). Expression of three major Mal d 1 isoforms was observed by real-time polymerase chain reaction over the 5 month storage period, and Mal d 1.02 was the most highly expressed isoform. In conclusion, Mal d 1 gene expression was significantly increased during modified atmosphere storage. Individuals suffering from birch pollen-apple allergy syndrome might experience fewer problems consuming freshly picked apples.

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[77] - Sancho AI, Foxall R, Rigby NM, Browne T, Zuidmeer L, van Ree R, et al. Maturity and storage influence on the apple (Malus domestica) allergen Mal d 3, a nonspecific lipid transfer protein. J Agric Food Chem 2006;54:5098-5104

Consumption of apples can provoke severe allergic reactions, in susceptible individuals, due to the presence of the allergen Mal d 3, a nonspecific lipid transfer protein, found largely in the fruit skin. Levels of Mal d 3 were determined in peel as a function of apple cultivar, position of the fruit growing on the tree, apple maturity, and postharvest storage by ELISA. As the apples mature, Mal d 3 levels increased, although the rate was dependent on cultivar and tree position. During storage, levels of Mal d 3 decreased in all cultivars (cvs. Cox, Jonagored, and Gala), the rate of overall decrease being greatest under controlled atmosphere conditions. There was no correlation between Mal d 3 levels and total apple peel protein, indicating specific alterations in Mal d 3 expression. Thus pre- and postharvest treatments (i.e., storage) can modify the allergen load in apple peel, the highest levels being found in overly mature and freshly harvested fruits.

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[78] - Bolhaar STHP, van de Weg WE, van Ree R, Gonzalez-Mancebo E, Zuidmeer L, Bruijnzeel-Koomen CAFM, et al. In vivo assessment with prick-to-prick testing and double-blind, placebo-controlled food challenge of allergenicity of apple cultivars. J Allergy Clin Immunol 2005;116:1080-1086

BACKGROUND: Apple cultivars have been reported to differ in allergenicity on the basis of in vitro and skin prick tests with apple extracts . OBJECTIVES: We sought to evaluate the efficacy of the prick-to-prick method in assessing differences in allergenicity of apple cultivars and to confirm differences by means of double-blind, placebo-controlled food challenge (DBPCFC) . METHODS: Intra-assay and intracultivar variation of prick-to-prick test results were determined in 6 Dutch and 8 Spanish patients with apple allergy by using 5 apples of the cultivars Golden Delicious, Fuji, and Ecolette in duplicate. In addition, 21 cultivars were screened for allergenicity in 15 Dutch patients with birch pollen and apple allergy. Two selected cultivars (Golden Delicious and Santana) were tested with DBPCFCs. The influence of storage conditions on allergenicity was assessed in 5 cultivars . RESULTS: Intra-assay variation of skin prick testing was 3.9%, and intracultivar variation was 4.1%. A ranking of 21 cultivars was made on the basis of prick-to-prick tests in 9 patients. Apple cultivars were classified as of low, intermediate, and high allergenicity, with a significant difference between low and high allergenicity (P < .001). A significant difference in allergenicity determined between Golden Delicious and Santana cultivars (P < .05) was confirmed by means of DBPCFC. With 5 cultivars, controlled atmosphere (2.5% oxygen/1% carbon dioxide) was shown to reduce allergenicity (P < .001) by 15% compared with storage at 2 degrees C . CONCLUSIONS: Prick-to-prick testing with fresh apples is a reproducible method of assessing allergenicity. Apples can be classified as of low or high allergenicity for the majority of patients. This was confirmed by using DBPCFCs. Selection of cultivars and control of storage conditions are both viable strategies for reduction of symptoms in patients with apple allergy.

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[79] - Santaolalla M, Baeza ML, Zapatero L, Frutos C, Rodriguez A, Zubeldia J, et al. Tolerance to Apple after Microwave Heating. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°1136

BACKGROUND: Heating of fruits can reduce allergenicity or create new allergens, but there are few references about microwave (MW) heating. METHODS: Skin prick tests (SPT) with both fresh apple and apple after 2 minutes in MW at 800W were made. Total IgE and apple-specific IgE were measured. In two of the patients we made an ELISA and an Immunoblot after making two extracts of apple in our laboratory: with fresh and with MW heated apple. Oral challenge tests with fresh and with MW heated apple were made in four of the patients. RESULTS: In all patients SPT were positive to fresh apple and negative to MW heated apple. Total IgE was 200, 365, 419, 721 and 2504 kU/l respectively. Apple-specific IgE by CAP-System was 0.96,2.2, 5.6, 4.6 and 8.6 kU/l respectively. ELISA was positive with fresh extract and negative with the MW heated extract in the two patients. Immunoblot revealed two IgE-binding proteins, at 42 and 36 kd with MW heated apple extract and only the 42 kd one with fresh apple extract. The four patients had symptoms after fresh apple intake but tolerated MW heated apple. CONCLUSIONS: (1) Two minutes of MW heating is a single and quick method that can let many allergy patients tolerate apples. (2) SPT with MW heated fruits seems to be a diagnostic tool and can predict tolerance before oral challenge test. (3) The 36 kd IgE-binding protein found by immunoblot in MW heated apple extract could be a neo allergen without clinical relevance.

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[80] - Asero R, Mistrello G, Roncarolo D, Amato S. Prick test with heat-processed apple peel extract to detect LTP hypersensitivity. Eur Ann Allergy Clin Immunol 2006;38:351-354

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.

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[81] - Smole U, Bublin M, Radauer C, Ebner C, Breiteneder H. Mal d 2, the Thaumatin-Like Allergen from Apple, Is Highly Resistant to Gastrointestinal Digestion and Thermal Processing. Int Arch Allergy Immunol 2008;147:289-298

BACKGROUND: The stability of food allergens to proteolysis and thermal denaturation contributes considerably to their allergenicity . METHODS: Mal d 2, an allergenic thaumatin-like protein (TLP) from apple, was isolated and purified by anion exchange chromatography. Its IgE reactivity was tested by ELISA and immunoblotting using sera from apple allergic patients. The proteolytic stability of Mal d 2 was investigated using two in vitro digestion models. Effects of thermal treatment at different pH values on the secondary structure of the protein were recorded by circular dichroism. The level and IgE reactivity of Mal d 2 present in industrially prepared foods were assessed . RESULTS: Purified Mal d 2 consisted of two isoforms. Both harbored carbohydrate moieties and bound serum IgE from apple allergic individuals. Mal d 2 showed remarkable stability to proteolysis and thermal treatments. The allergen remained intact after 2 h each of gastric and subsequent duodenal digestion retaining its full IgE-binding capacity. Mal d 2 was unfolded at neutral and acidic pH at 70 degrees C. Refolding after cooling was only observed at acidic pH. Mal d 2 detected by an anti-TLP antibody in cloudy apple juice did not bind IgE of a serum pool of apple allergic patients . CONCLUSION: Our findings suggest that Mal d 2 maintains its structure in the gastrointestinal tract, a feature essential for sensitizing the mucosal immune system and provoking allergic reactions.

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[82] - Skamstrup Hansen K, Vestergaard H, Stahl Skov P, Sùndergaard Khinchi M, Vieths S, Poulsen LK, et al. Double-blind, placebo-controlled food challenge with apple. Allergy 2001;56:109-117

The aim of the study was to develop and evaluate different methods of double-blind, placebo-controlled food challenge (DBPCFC) with apple. Three different DBPCFC models were evaluated: fresh apple juice, freshly grated apple, and freeze-dried apple powder. All challenges were performed outside the pollen season and took place from 1997 to 1999. The freeze-dried apple material was characterized by means of leukocyte histamine release (HR), skin prick test (SPT), and immunoblotting experiments. The study population consisted of birch pollen-allergic patients with a history of rhinitis in the birch-pollen season and positive specific IgE to birch. For comparison of the DBPCFC models, 65 patients with a positive open oral challenge with apple were selected. In the characterization of the freeze-dried apple material, 46 birch pollen-allergic patients were included. The IgE reactivity to apple was evaluated by measurement of specific IgE, HR, and SPT. Golden Delicious apples were used in all experiments. The results of this study showed that it was possible to perform DBPCFC with apple in birch pollen-allergic individuals. The model with freshly squeezed apple juice had a low sensitivity and displayed a high frequency of reactions to placebo, probably due to the ingredients used for blinding. The sensitivity of the models with freshly grated apple and freeze-dried apple powder was 0.74/0.60. An increase in sensitivity is desirable. The freeze-dried apple powder proved to be useful for SPT, HR, and oral challenges, but further investigation of the stability and the allergenic profile of the material is needed.

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[83] - Smole U, Bublin M, Radauer C, Ebner C, Breiteneder H. Mal d 2, the Thaumatin-Like Allergen from Apple, Is Highly Resistant to Gastrointestinal Digestion and Thermal Processing. Int Arch Allergy Immunol 2008;147:289-298

BACKGROUND: The stability of food allergens to proteolysis and thermal denaturation contributes considerably to their allergenicity . METHODS: Mal d 2, an allergenic thaumatin-like protein (TLP) from apple, was isolated and purified by anion exchange chromatography. Its IgE reactivity was tested by ELISA and immunoblotting using sera from apple allergic patients. The proteolytic stability of Mal d 2 was investigated using two in vitro digestion models. Effects of thermal treatment at different pH values on the secondary structure of the protein were recorded by circular dichroism. The level and IgE reactivity of Mal d 2 present in industrially prepared foods were assessed . RESULTS: Purified Mal d 2 consisted of two isoforms. Both harbored carbohydrate moieties and bound serum IgE from apple allergic individuals. Mal d 2 showed remarkable stability to proteolysis and thermal treatments. The allergen remained intact after 2 h each of gastric and subsequent duodenal digestion retaining its full IgE-binding capacity. Mal d 2 was unfolded at neutral and acidic pH at 70 degrees C. Refolding after cooling was only observed at acidic pH. Mal d 2 detected by an anti-TLP antibody in cloudy apple juice did not bind IgE of a serum pool of apple allergic patients . CONCLUSION: Our findings suggest that Mal d 2 maintains its structure in the gastrointestinal tract, a feature essential for sensitizing the mucosal immune system and provoking allergic reactions.

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[84] - Asero R, Mistrello G, Roncarolo D, Amato S, Falagiani P. Analysis of the heat stability of lipid transfer protein from apple. J Allergy Clin Immunol 2003;112:1009-1011

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[85] - Sancho AI, Rigby NM, Zuidmeer L, Asero R, Mistrello G, Amato S, et al. The effect of thermal processing on the IgE reactivity of the non-specific lipid transfer protein from apple, Mal d 3. Allergy 2005;60:1262-1268

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are involved in allergy to fresh and processed fruits. We have investigated the effect of thermal treatment and glycation on the physico-chemical and IgE-binding properties of the LTP from apple (Mal d 3) . METHODS: Mal d 3 was purified from apple peel and the effect of heating in the absence and presence of glucose investigated by CD spectroscopy, electrospray and MALDI-TOF mass spectrometry. IgE reactivity was determined by RAST and immunoblot inhibition, SPT and basophil histamine release test . RESULTS: The identity and IgE reactivity of purified Mal d 3 was confirmed. Mild heat treatment (90 degrees C, 20 min) in the absence or presence of glucose did not alter its IgE reactivity. More severe heat treatment (100 degrees C, 2 h) induced minor changes in protein structure, but a significant decrease in IgE-binding (30-fold) and biological activity (100- to 1000-fold). Addition of glucose resulted in up to four glucose residues attached to Mal d 3 and only a 2- and 10-fold decrease of IgE-binding and biological activity, respectively . CONCLUSIONS: Only severe heat treatment caused a significant decrease in the allergenicity of Mal d 3 but glycation had a protective effect. The presence of sugars in fruits may contribute to the thermostability of the allergenic activity of LTP in heat-processed foods.

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[86] - Polovic N, Blanusa M, Gavrovic-Jankulovic M, Atanaskovic-Markovic M, Burazer L, Jankov R, et al. A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro. Clin Exp Allergy 2007;37:764-771

Abstract Background It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low-pH conditions. Objective The purpose of this study was to investigate the effect of hydrogel forming polysaccharide-rich fruit matrixes on in vivo gastric and in vitro pepsic digestion of fruit allergens. Methods Fruit extract proteins (kiwi, banana, apple and cherry) and a purified major kiwi allergen Act c 2 were digested with simulated gastric fluid in accordance with the US Pharmacopeia. In vivo experiments on kiwi fruit digestion were performed on four healthy non-atopic volunteers by examining the gastric content 1 h after ingestion of kiwi fruit. The Act c 2 and kiwi proteins were detected in immunoblots using monoclonal anti-Act c 2 antibodies and rabbit polyclonal antisera. Results Crude fruit extracts were resistant to digestion by pepsin when compared with commonly prepared extracts. In the gastric content of all volunteers, following kiwi fruit ingestion and immunoblotting, intact Act c 2 was detected with anti-Act c 2 monoclonal antibodies, while kiwi proteins of higher molecular weights were detected using rabbit polyclonal antisera. Addition of apple fruit pectin (1.5% and 3%) to the purified kiwi allergen was able to protect it from pepsin digestion in vitro. Conclusion The matrix effect in pectin-rich fruits can influence the digestibility of food proteins and thereby the process of allergic sensitization in atopic individuals.

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[87] - Smole U, Bublin M, Radauer C, Ebner C, Breiteneder H. Mal d 2, the Thaumatin-Like Allergen from Apple, Is Highly Resistant to Gastrointestinal Digestion and Thermal Processing. Int Arch Allergy Immunol 2008;147:289-298

BACKGROUND: The stability of food allergens to proteolysis and thermal denaturation contributes considerably to their allergenicity . METHODS: Mal d 2, an allergenic thaumatin-like protein (TLP) from apple, was isolated and purified by anion exchange chromatography. Its IgE reactivity was tested by ELISA and immunoblotting using sera from apple allergic patients. The proteolytic stability of Mal d 2 was investigated using two in vitro digestion models. Effects of thermal treatment at different pH values on the secondary structure of the protein were recorded by circular dichroism. The level and IgE reactivity of Mal d 2 present in industrially prepared foods were assessed . RESULTS: Purified Mal d 2 consisted of two isoforms. Both harbored carbohydrate moieties and bound serum IgE from apple allergic individuals. Mal d 2 showed remarkable stability to proteolysis and thermal treatments. The allergen remained intact after 2 h each of gastric and subsequent duodenal digestion retaining its full IgE-binding capacity. Mal d 2 was unfolded at neutral and acidic pH at 70 degrees C. Refolding after cooling was only observed at acidic pH. Mal d 2 detected by an anti-TLP antibody in cloudy apple juice did not bind IgE of a serum pool of apple allergic patients . CONCLUSION: Our findings suggest that Mal d 2 maintains its structure in the gastrointestinal tract, a feature essential for sensitizing the mucosal immune system and provoking allergic reactions.

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[88] - Pastorello EA, Conti A, Pravettoni V, Farioli L, Rivolta F, Ansaloni R, et al. Identification of actinidin as the major allergen of kiwi fruit. J Allergy Clin Immunol 1998;101:531-537

Allergic reactions to fruits and vegetables are among the most frequent food allergies in adults. Kiwi fruit (Actinidia chinensis) is commonly involved, causing local mucosal, systemic, or both types of symptoms by an IgE-mediated mechanism. In a previous study on 30 patients allergic to kiwi, we identified a major allergen of 30 kd against which all sera tested clearly reacted. Other allergens were detected at 12, 24, and 28 kd. OBJECTIVE: The aim of this study was to fully characterize the major kiwi fruit allergen of 30 kd. METHODS: Allergens were separated and purified by high-performance liquid chromatography with anion-exchange columns. The purity of the single proteins was checked by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and their allergenicity was checked by immunoblotting with a pool of sera from patients allergic to kiwi. The allergens were characterized by isoelectrofocusing and amino acid sequencing, and periodic acid-Schiff stain was used to detect glycoproteins. RESULTS: Proteins of 30, 28, 24, and 17 kd were purified by high-performance liquid chromatography. IgE binding indicated the 30 kd protein, which showed an isoelectric point of 3.5, as the major allergen of kiwi. Determination of its partial amino acid sequence and comparison with the Swiss Protein Bank showed that this was actinidin, the main protein component of kiwi. The 24 and 28 kd proteins had the same N-terminal sequence, which did not correspond to any known protein. The 17 kd protein had a blocked N-terminal sequence. CONCLUSIONS: These results demonstrate that the major allergen of kiwi fruit, Act c 1, is actinidin, a proteolytic enzyme belonging to the class of thiol-proteases. Two other allergens of 24 and 28 kd appear identical on amino acid sequencing.

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[89] - Akkerdaas JH, Wensing M, Asero R, Fernandez Rivas M, Knulst AC, Bolhaar S, et al. IgE Binding to Pepsin-Digested Food Extracts. Int Arch Allergy Immunol 2005;138:203-208

BACKGROUND: Pepsin resistance of allergens like lipid transfer protein and 2S albumin has been suggested as explanation for the severity of symptoms often induced by these allergens. Component-resolved diagnosis with purified labile and stable allergens has therefore been proposed to better characterize the risk involved in a positive in vitro IgE test. However, for many foods, purified allergens are not (yet) available. OBJECTIVE: It was the aim of this study to evaluate the potential of pepsin-digested whole-food extracts to distinguish between IgE responses to stable (potentially severe) and labile (mild) allergens. METHODS: Sera (n = 143) from Italian, Spanish and Dutch patients with hazelnut and/or apple ingestion-related symptoms were analyzed for residual IgE binding to pepsin-resistant hazelnut and/or apple allergens. Control and pepsin-digested hazelnut and apple extracts were used for radioallergosorbent test analysis and immunoblot analysis. RESULTS: Pepsin digestion of food extracts, like from hazelnut and apple used for in vitro diagnostic tests, provides a way to distinguish sensitization to pepsin-resistant allergens from that to pepsin-susceptible allergens. In this selected group of patients, IgE reactivity to pepsin-digested extracts correlated with sensitization to the stable allergen lipid transfer protein. The analysis further revealed that the use of soluble pepsin can result in false-positive in vitro tests (2/143). CONCLUSION: Pepsin-digested food extracts are a convenient tool to identify patients with IgE antibodies against potentially dangerous stable allergens, in particular for those foods where the relevant stable allergens have not yet been identified. This can increase the clinical prognostic value of food allergy serology.

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Les allergènes de la pomme

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