Le bouleau

Positifs pour	Sujets (%)
	Mono bouleau	Bouleau + autres pollens
Bet v 1	8	6
Bet v 1 et Bet v 2	1	29
Bet v 2	0	6

	Bet v 1	Bet v 2	Bet v 4
Finlande	100	2	5
Suède	98	12	8
Allemagne	89 / 91	13 / 18	6
Pays-Bas	85	19
Suisse	65 / 92	43 / 24	7 / 2
Autriche	98	30 / 18	11
Belgique		21
France	90 / 74	20 / 20	6
Italie (Nord)	62 / 79	33 / 30 / 57	27

% de mono-réactivité parmi les positifs pour
	rBet v 1	r Bet v 2	r Bet v 4
Finlande	92	0	0
Suède	86	0	0
Autriche	67	2	0
France	80	4	2
Suisse	46	11	4
Italie	46	27	0

[1] - Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, et al. The gene coding for the major birch pollen allergen, Bet v 1, is highly homologous to a pea disease resistance response gene. EMBO J 1989;8:1935-1938

Pollen of the white birch (Betula verrucosa) is one of the main causes of Type I allergic reactions (allergic rhinoconjunctivitis, allergic bronchial asthma) in Middle and Northern Europe, North America and the USSR. Type I allergies are a major threat to public health in these countries, since 10-15% of the population suffer from these diseases. BetvI, an allergenic protein with an Mr of 17 kd is a constituent of the pollen of white birch and is responsible for IgE binding in more than 95% of birch pollen allergic patients. Here, we report the complete nucleotide sequence and deduced amino acid sequence of a cDNA clone coding for the major pollen allergen (BetvI) of white birch. It is similar to the N-terminal peptide sequences of the allergens of hazel, alder and hornbeam (close relatives) but it has no significant sequence homology to any other known allergens. However, it shows 55% sequence identity with a pea disease resistance response gene, indicating that BetvI may be involved in pathogen resistance of pollen.

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[2] - Valenta R, Duchêne M, Pettenburger K, Sillaber C, Valent P, Bettelheim P, et al. Identification of profilin as a novel pollen allergen: IgE autoreactivity in sensitized individuals. Science 1991;253:557-560

A complementary DNA encoding a pollen allergen from white birch (Betula verrucosa) that was isolated from a pollen complementary DNA library with serum immunoglobulin E from a birch pollen-allergic individual revealed significant sequence homology to profilins. The recombinant protein showed high affinity to poly-L-proline. Immunoglobulin E antibodies from allergic individuals bound to natural and recombinant birch profilin and also to human profilin. In addition, birch and human profilin induced histamine release from blood basophils of profilin-allergic individuals, but not of individuals sensitized to other plant allergens. The structural similarity of conserved proteins might therefore be responsible for maintaining immunoglobulin E antibody titers in type I allergy.

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[3] - Valenta R, Lidholm J, Niederberger V, Hayek B, Kraft D, Grönlund H. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 1999;29:896-904

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[4] - 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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[5] - Pauli G, Oster JP, Deviller P, Heiss S, Bessot JC, Susani M, et al. Skin testing with recombinant allergens rBet v 1 and birch profilin, rBet v 2: diagnostic value for birch pollen and associated allergies. J Allergy Clin Immunol 1996;97:1100-1109

This study assesses the value of two recombinant birch allergens for diagnosis of patients sensitized to birch pollen with or without associated food allergy. METHODS: Fifty-one patients with positive skin test responses to Betulaceae and seven nonallergic control subjects were investigated; specific IgE antibodies were evaluated by specific immunoassay and blot immunodetection. RESULTS: Among 51 patients, 47 reacted to rBet v 1 and 10 to rBet v 2. Seven patients reacted to both recombinant allergens. In skin prick tests we found a correlation between the wheal produced by the commercial birch extract and the wheal produced by rBet v 1. Among 47 patients with positive test responses to rBet v 1, 83% had IgE binding to the Bet v 1 protein as determined by immunoblotting. Among 10 patients sensitized to rBet v 2, six had IgE binding to Bet v 2. Eleven patients with negative results, as determined by immunoblotting, had low levels of birch IgE in the sera (less than 10 kU/L) and low concentrations of IgE to rBet v 1 or rBet v 2 in ELISA. The nonallergic control subjects (n = 7) did not react to rBet v 1 or rBet v 2 in skin prick tests, nor did they have detectable amounts of specific IgE to rBet v 1 or rBet v 2. Histamine release tests confirmed sensitization to Bet v 1 in two patients with discordant results; for Bet v 2, one patient had positive results only at a high concentration, and one had results that remained negative. Thirty-four patients had birch pollinosis, and all reacted to rBet v 1. Patients who were monosensitized to birch never reacted to rBet v 2. Sensitization to rBet v 2 was only found in patients who reacted to other pollens (mainly grass). Twenty-nine patients demonstrated allergy to apples, cherries, or hazelnuts; and all reacted to rBet v 1. Among 11 patients with allergy to Umbelliferae, only three reacted to rBet v 2. CONCLUSIONS: Use of the two recombinant allergens (rBet v 1 and rBet v 2) always permits the diagnosis of birch sensitization. Sensitization to rBet v 1 is specific for birch and Rosaceae allergies, whereas sensitization to birch profilin, Bet v 2, is encountered in multisensitized subjects and is not always related to Umbelliferae allergy.

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[6] - Niederberger V, Reisinger J, Valent P, Krauth MT, Pauli G, van Hage M, et al. Vaccination with genetically modified birch pollen allergens: Immune and clinical effects on oral allergy syndrome. J Allergy Clin Immunol 2007;119:1013-1016

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[7] - Valenta R, Duchene M, Vrtala S, Birkner T, Ebner C, Hirschwehr R, et al. Recombinant allergens for immunoblot diagnosis of tree-pollen allergy. J Allergy Clin Immunol 1991;88:889-894

Diagnosis of type I allergy essentially depends on the availability of defined allergens, which can be provided by recombinant deoxyribonucleic acid (DNA) technology. We have previously isolated the c(complementary)DNAs encoding the major birch-pollen allergen, Bet v I, and another allergen with a molecular weight of 14 kd that was identified as birch profilin and designated Bet v II. These cDNAs were isolated from a lambda gt11 expression library by screening with the serum IgE from allergic patients. To obtain expression in Escherichia coli of recombinant allergens without additional fused polypeptides, both cDNAs were inserted into the plasmid pKK223-3. E. coli cells expressing Bet v I and birch profilin (Bet v II) were used for the preparation of recombinant proteins. These proteins were tested for their IgE-binding properties on immunoblots with sera from 100 different birch pollen-allergic patients. All patients' sera, which reacted with the natural allergens, Bet v I and Bet v II, demonstrated an ident ical IgE-binding pattern to recombinant birch-pollen allergens. Recombinant allergens may therefore be useful for the setup of diagnostic tests that allow the discrimination of different IgE-binding patterns as well as for patient-tailored immunotherapy

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[8] - 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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[9] - 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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[10] - Ipsen H, Lowenstein H. Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa). J Allergy Clin Immunol 1983;72:150-159

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest "radiostaining," and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing

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[11] - Schenk MF, Gilissen LJ, Esselink GD, Smulders MJ. Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen. BMC Genomics 2006;7:168

ABSTRACT: BACKGROUND: Pollen of the European white birch (Betula pendula, syn. B. verrucosa) is an important cause of hay fever. The main allergen is Bet v 1, member of the pathogenesis-related class 10 (PR-10) multigene family. To establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree, PCR amplification, cloning and sequencing of PR-10 genes was performed on two diploid B. pendula cultivars and one interspecific tetraploid Betula hybrid. Sequences were attributed to putative genes based on sequence identity and intron length. Information on transcription was derived by comparison with homologous cDNA sequences available in GenBank/EMBL/DDJB. PCR-cloning of multigene families is accompanied by a high risk for the occurrence of PCR recombination artifacts. We screened for and excluded these artifacts, and also detected putative artifact sequences among database sequences. RESULTS: Forty-four different PR-10 sequences were recovered from B. pendula and assigned to thirteen putative genes. Sequence homology suggests that three genes were transcribed in somatic tissue and seven genes in pollen. The transcription of three other genes remains unknown. In total, fourteen different Bet v 1-type isoforms were identified in the three cultivars, of which nine isoforms were entirely new. Isoforms with high and low IgE-reactivity are encoded by different genes and one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. Allergen diversity is even higher in the interspecific tetraploid hybrid, consistent with the presence of two genomes. CONCLUSIONS: Isoforms of the major birch allergen Bet v 1 are encoded by multiple genes, and we propose to name them accordingly. The present characterization of the Bet v 1 genes provides a framework for the screening of specific Bet v 1 genes among other B. pendula cultivars or Betula species, and for future breeding for trees with a reduced allergenicity. Investigations towards sensitization and immunotherapy should anticipate that patients are exposed to a mixture of Bet v 1 isoforms of different IgE-reactivity, even if pollen originates from a single birch tree.

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[12] - Wagner S, Radauer C, Bublin M, Hoffmann-Sommergruber K, Kopp T, Greisenegger EK, et al. Naturally occurring hypoallergenic Bet v 1 isoforms fail to induce IgE responses in individuals with birch pollen allergy. J Allergy Clin Immunol 2008;121:246-252

BACKGROUND: Engineered hypoallergens are currently being investigated for specific immunotherapy of allergic diseases in preclinical and clinical studies. Naturally occurring hypoallergens have by and large not been considered as a source of vaccine candidates . OBJECTIVE: Evaluation of the antibody response in atopic individuals induced by birch pollen containing isoforms of the major birch pollen allergen Bet v 1 . METHODS: Isoform-specific antibody isotype responses for Bet v 1.0101, Bet v 1.0401, and Bet v 1.1001 were determined for 35 sera of individuals with birch pollen allergy. Isoform structures were compared and related to IgE-binding inhibitory capacities and induction of mediator release in human Fcvarepsilon receptor transformed rat basophilic leukemia cells . RESULTS: Bet v 1.0101 induced a predominant IgE response, whereas the significant highest levels of IgG(4) antibodies were directed against Bet v 1.0401. Bet v 1.1001 induced only a minimal antibody response. Structural comparisons revealed that most of the amino acid differences between the isoforms were located on the protein surfaces. IgE induced by Bet v 1.0101 only partly cross-reacted with the 2 other isoforms and bound to them with notably lower affinity. Bet v 1.0401 and Bet v 1.1001 also were poor inducers of mediator release . CONCLUSION: Bet v 1 isoforms possess highly variant immunogenic and allergenic properties. Bet v 1.0101 acts as the sensitizing agent, whereas Bet v 1.0401 and Bet v 1.1001 can induce only a minimal IgE response.

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[13] - Schenk MF, Cordewener JH, America AH, Van't Westende WP, Smulders MJ, Gilissen LJ. Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen. BMC Plant Biol 2009;3(9):24

ABSTRACT: BACKGROUND: Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. RESULTS: All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives. CONCLUSION: Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.

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[14] - Malandain H, Lavaud F. Allergénicité des protéines de défense végétale. Rev Fr Allergol Immunol Clin 2004;44:469-475

La synthèse de protéines de défense fait partie des réponses biochimiques que les plantes ont développées pour lutter contre leurs ennemis naturels et contre les stress environnementaux. De nombreuses protéines de défense végétale sont allergisantes : chitinases, protéines de transfert lipidique LTP, protéines Bet v 1-like. Cet article rappelle les principales familles de protéines de défense végétale, leur impact allergique et l'effet de certains procédés agricoles ou technoalimentaires sur l'allergénicité des aliments végétaux.

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[15] - 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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[16] - Menz G, Dolecek C, Schonheit-Kenn U, Ferreira F, Moser M, Schneider T, et al. Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen. Clin Exp Allergy 1996;26:50-60

BACKGROUND: Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer from allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis, leading to death. OBJECTIVE: The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy. METHODS: A group of 51 birch pollen allergic patients and eight non- allergic control individuals were tested for birch pollen allergy by skin-prick and intradermal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained i n any in vitro assay system (ELISA, Western blot) or by skin testing (skin- prick, intradermal test) with recombinant Bet v I. CONCLUSION: Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients

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[17] - Purohit A, Laffer S, Niederberger V, Lieutier-Colas F, De Blay F, Valenta R, et al. The Discrepancy Between Quantitative IgE Serology, Skin Test Sensitivity and Basophil Releasability Analysed With Recombinant Bet v 1, the Major Birch Pollen Allergen. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°309

BACKGROUND: Using allergen extracts for allergy diagnosis, discrepancies between clinical sensitivity determined by cutaneous and basophil provocation and specific serum IgE levels have frequently been reported. This observation may be due to several factors including I) the presence of varying amounts of highly or less allergenic molecules within allergen extracts, II) different effector cell sensitivity and III) effects of antibodies other than IgE on these determinations. AIM: To compare skin test sensitivity, allergen-specific IgE levels and basophil histamine releasability in birch pollen allergic patients using a purified, defined and structurally folded recombinant allergen, rBet v 1. METHODS: Threshold endpoint intradermal skin tests were performed by injection of 0.03 ml of 10-fold dilutions of rBet 1 in 18 birch pollen allergic patients. rBet v 1-specific IgE were measured by quantitative CAP-RAST determinations and IgG1-4 subclass responses by ELISA. Basophil histamine release assays were done with rBet v 1. For each patient all assays were performed on the same day before the birch pollen season. RESULTS: rBet v 1-specific IgE levels ranged from 1.65 kUA/L to 79.9 kUA/L in the study group and the amount of rBet v 1 required to induce a positive intradermal reaction varied from 10-5 to 10 µg/ml. Concentrations of rBet v 1 required to induce 30% or maximal histamine release varied from 10-3 to 1 and 10-2 to 10 µg/ml respectively. There was no correlation between either skin test sensitivity and specific IgE levels (r= 0.27, p= 0.27) or between basophil releasability and specific IgE levels (30% histamine release: r= -0.27, p= 0.27; maximal histamine release: r= 0.05, p= 0.84). Modulating effects of allergen-specific antibodies other than IgE appeared unlikely because no rBet v 1-specific IgA or IgM could be detected in the sera and there was no obvious association of high rBet v 1 specific IgG subclass responses and low sensitivity. CONCLUSION: Even at the molecular level, skin test sensitivity or basophil releasability are not well correlated with allergen-specific IgE levels. Factors (e.g. epitope density/geometry, IgE heterogeneity) other than different effector cell sensitivity and modulating antibodies may be responsible for this observation

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[18] - Mothes-Luksch N, Spitzauer S, Hauswirth A, Krauth M, Valent P, Steiner R, et al. Comparison of in vivo allergenic activity of recombinant Bet v 1 with allergen-specific in vitro immune responses in birch pollen allergic patients. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1056

Background: There is a need for in vitro markers for allergic inflammation but several studies have demonstrated that allergen-specific in vitro immune responses are not always correlated with in vivo allergenic activity. Objective: To perform an extended analysis of in vitro immune responses (antibody, cytokine, basophil activation) with in vivo (skin prick test) allergenic activity to one defined allergen molecule in allergic patients. Methods: The in vivo allergenic activity of purified recombinant birch pollen allergen, Bet v 1, was studied in twenty birch pollen allergic patients who had not received immunotherapy and five non-allergic individuals by skin prick testing with serial (1:2) allergen dilutions. The analysis of in vitro immune responses included besides classical parameters (allergen-specific antibodies: IgE levels by CAP, IgE, IgG1-4 IgM and IgA by ELISA; allergen-induced basophil histamine release; Th2 and Th1 cytokine production in allergen-stimulated PBMCs: IL-4, IL-5, IFN-g) also the new basophil activation marker CD203c and the tolerogenic cytokine IL-10. Results: With the exception of one patient whose basophils did not respond to IgE-mediated stimulation and one patient without detectable Bet v 1- specific IgE, allergen-specific IgE reactivity, allergen-induced basophil activation (histamine release, CD203c) and skin responses were always associated but the magnitude of the individual responses was poorly correlated. This poor correlation could not be explained by the presence of natural Bet v 1-specific IgG or IgA antibodies nor by the levels of the Th1 cytokine IFN-g or the tolerogenic cytokine IL-10. The levels of IL-10 secreted by allergen-stimulated PBMC were even lower in the non-allergic individuals than in the allergic patients. IL-5 levels were higher in allergen-stimulated PBMCs from allergic patients than in PBMCs from the non-allergic individuals. Conclusion: Our study emphasizes that the assessment of clinical allergen-specific sensitivity cannot be fully replaced by in vitro parameters but requires in vivo testing.

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[19] - Mothes-Luksch N, Spitzauer S, Hauswirth A, Krauth M, Valent P, Steiner R, et al. Comparison of in vivo allergenic activity of recombinant Bet v 1 with allergen-specific in vitro immune responses in birch pollen allergic patients. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1056

Background: There is a need for in vitro markers for allergic inflammation but several studies have demonstrated that allergen-specific in vitro immune responses are not always correlated with in vivo allergenic activity. Objective: To perform an extended analysis of in vitro immune responses (antibody, cytokine, basophil activation) with in vivo (skin prick test) allergenic activity to one defined allergen molecule in allergic patients. Methods: The in vivo allergenic activity of purified recombinant birch pollen allergen, Bet v 1, was studied in twenty birch pollen allergic patients who had not received immunotherapy and five non-allergic individuals by skin prick testing with serial (1:2) allergen dilutions. The analysis of in vitro immune responses included besides classical parameters (allergen-specific antibodies: IgE levels by CAP, IgE, IgG1-4 IgM and IgA by ELISA; allergen-induced basophil histamine release; Th2 and Th1 cytokine production in allergen-stimulated PBMCs: IL-4, IL-5, IFN-g) also the new basophil activation marker CD203c and the tolerogenic cytokine IL-10. Results: With the exception of one patient whose basophils did not respond to IgE-mediated stimulation and one patient without detectable Bet v 1- specific IgE, allergen-specific IgE reactivity, allergen-induced basophil activation (histamine release, CD203c) and skin responses were always associated but the magnitude of the individual responses was poorly correlated. This poor correlation could not be explained by the presence of natural Bet v 1-specific IgG or IgA antibodies nor by the levels of the Th1 cytokine IFN-g or the tolerogenic cytokine IL-10. The levels of IL-10 secreted by allergen-stimulated PBMC were even lower in the non-allergic individuals than in the allergic patients. IL-5 levels were higher in allergen-stimulated PBMCs from allergic patients than in PBMCs from the non-allergic individuals. Conclusion: Our study emphasizes that the assessment of clinical allergen-specific sensitivity cannot be fully replaced by in vitro parameters but requires in vivo testing.

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[20] - Purohit A, Laffer S, Niederberger V, Lieutier-Colas F, De Blay F, Valenta R, et al. The Discrepancy Between Quantitative IgE Serology, Skin Test Sensitivity and Basophil Releasability Analysed With Recombinant Bet v 1, the Major Birch Pollen Allergen. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°309

BACKGROUND: Using allergen extracts for allergy diagnosis, discrepancies between clinical sensitivity determined by cutaneous and basophil provocation and specific serum IgE levels have frequently been reported. This observation may be due to several factors including I) the presence of varying amounts of highly or less allergenic molecules within allergen extracts, II) different effector cell sensitivity and III) effects of antibodies other than IgE on these determinations. AIM: To compare skin test sensitivity, allergen-specific IgE levels and basophil histamine releasability in birch pollen allergic patients using a purified, defined and structurally folded recombinant allergen, rBet v 1. METHODS: Threshold endpoint intradermal skin tests were performed by injection of 0.03 ml of 10-fold dilutions of rBet 1 in 18 birch pollen allergic patients. rBet v 1-specific IgE were measured by quantitative CAP-RAST determinations and IgG1-4 subclass responses by ELISA. Basophil histamine release assays were done with rBet v 1. For each patient all assays were performed on the same day before the birch pollen season. RESULTS: rBet v 1-specific IgE levels ranged from 1.65 kUA/L to 79.9 kUA/L in the study group and the amount of rBet v 1 required to induce a positive intradermal reaction varied from 10-5 to 10 µg/ml. Concentrations of rBet v 1 required to induce 30% or maximal histamine release varied from 10-3 to 1 and 10-2 to 10 µg/ml respectively. There was no correlation between either skin test sensitivity and specific IgE levels (r= 0.27, p= 0.27) or between basophil releasability and specific IgE levels (30% histamine release: r= -0.27, p= 0.27; maximal histamine release: r= 0.05, p= 0.84). Modulating effects of allergen-specific antibodies other than IgE appeared unlikely because no rBet v 1-specific IgA or IgM could be detected in the sera and there was no obvious association of high rBet v 1 specific IgG subclass responses and low sensitivity. CONCLUSION: Even at the molecular level, skin test sensitivity or basophil releasability are not well correlated with allergen-specific IgE levels. Factors (e.g. epitope density/geometry, IgE heterogeneity) other than different effector cell sensitivity and modulating antibodies may be responsible for this observation

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[21] - Niederberger V, Stubner P, Spitzauer S, Kraft D, Valenta R, Ehrenberger K, et al. Skin test results but not serology reflect immediate type respiratory sensitivity: a study performed with recombinant allergen molecules. J Investig Dermatol 2001;117:848-851

The diagnosis of type I allergy, an IgE-antibody-mediated hypersensitivity disease affecting more than 25% of the population, is based on the measurement of allergen-specific serum IgE levels and provocation testing. Whether the determination of allergen- specific serum IgE levels can replace in vivo provocation testing for allergy diagnosis is a controversial issue. We used purified recombinant timothy grass and birch pollen allergens to compare by skin prick and nasal provocation testing as well as by serology in vivo sensitivity with antibody-binding capacity in 24 pollen allergic patients and eight control individuals. Results from biologic tests were correlated with each other and with allergen-specific IgE and IgG1-4 levels. IgE-reactive allergens induced immediate skin and nasal reactions, but the intensity of the allergic tissue reactions was not correlated with either the levels of allergen-specific IgE or the levels of allergen-specific IgG antibodies. Less frequently detected allergens with low IgE-binding capacity were able to induce strong allergic reactions comparable to those caused by major allergens with high IgE-binding capacity. In contrast, skin test and nasal provocation results were significantly correlated (r = 0.63, p < 0.01). Our study thus demonstrates on a molecular level that skin testing provides a better reflection of immediate type respiratory sensitivity than serologic measurements. These results have implications for allergy diagnosis and, in particular, for the selection of relevant allergen components for specific immunotherapy.

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[22] - Tresch S, Holzmann D, Baumann S, Blaser K, Wüthrich B, Crameri R, et al. In vitro and in vivo allergenicity of recombinant Bet v 1 compared to the reactivity of natural birch pollen extract. Clin Exp Allergy 2003;33:1153-1158

BACKGROUND : Diagnostic procedures using natural extracts show only limited quantitative correlation between in vivo and in vitro results. Highly pure recombinant allergens might show more predictive findings. OBJECTIVE : The aim of this study was to compare natural birch pollen extract (BPE) and recombinant Betula verrucosa (rBet v 1) for their diagnostic value comparing skin prick tests (SPTS) and nasal provocation tests (NPTS) with specific IgE in the serum. METHODS : Thirty-four patients allergic to birch pollen and five healthy controls were investigated. SPT and NPT were performed with BPE and rBet v 1 at different concentrations. Specific serum IgE was measured by the Pharmacia CAP system. RESULTS : Commercial BPE and rBet v 1 (10 micro g/mL) were able to elicit similar allergenic reactions in vivo and IgE binding in vitro. SPT reflects immediate-type allergy as determined by NPT to a higher degree than specific IgE, for both reagents. To cause allergic reactions in NPT, higher amounts of rBet v 1 were needed than for skin tests and the sensitivity was lower than with BPE. CONCLUSION : rBet v 1 alone is sufficient for a reliable diagnosis of birch pollen allergy in most patients and induces comparable skin test reactivity as BPE, but less allergic reactions in nasal provocations.

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[23] - Wellhausen A, Schöning B, Petersen A, Vieths S. IgE binding to a new cross-reactive structure: a 35 kDa protein in birch pollen, exotic fruit and other plant foods. Z Ernahrungswiss 1996;35:348-355

Food allergies in birch pollen allergic patients have been shown to be due to cross-reactivities of specific IgE antibodies which are directed against birch pollen allergens with related proteins in fruit, nuts and vegetables. We identified a new cross-reactive structure of 35 kDa in birch pollen and some plant food extracts by Enzyme Allergosorbent Test (EAST) and immunoblot inhibition studies. The 35 kDa birch pollen protein is a minor allergen to which approximately 10-15% of birch pollen allergic individuals have specific IgE. Our data demonstrate that there is cross-reactivity of this protein with proteins of comparable size from lychee, mango, banana, orange, apple, pear and carrot. While the 35 kDa protein is immunologically independent of the major birch pollen allergen Bet v 1, we also observed IgE binding to a 34 kDa structure which appears to be a Bet v 1 dimer.

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[24] - Schöll I, Kalkura N, Shedziankova Y, Bergmann A, Verdino P, Knittelfelder R, et al. Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice. J Immunol 2005;175:6645-6650

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

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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] - Schocker F, Lüttkopf D, Scheurer S, Petersen A, Cisteró-Bahíma A, Enrique E, et al. Hazelnut LTP, Cor a 8: Molecular Cloning and Clinical Relevance. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°925

BACKGROUND: In Northern Europe the birch pollen-associated allergens Cor a 1.0401 and Cor a 2 (profilin) are known as cross-reactive allergens in hazelnuts causing milder allergic reactions such as the oral allergy syndrome (OAS). Recently, lipid transfer proteins (LTP) have been described mainly in the Mediterranean area as food allergens which often elicit more severe reactions due to their high stability. The aim of this study was to determine the primary structure of the LTP of hazelnut and to investigate its IgE prevalence in a group of Spanish patients suffering from hazelnut allergy and plane and/or grass pollinosis. The IgE-reactivity was compared to 2 German sera from patients with pollen-independent hazelnut allergy. Hazelnut LTP was further analysed for cross-reactivity to pollens. METHODS: On the basis of known homologous sequences of cDNAs of lipid transfer proteins a PCR strategy was chosen to obtain the full-length nucleotide sequence of hazelnut LTP. IgE reactivity of recombinant hazelnut LTP, rCor a 1.0401, and rCor a 2 was tested by EAST in the Spanish patients and the 2 German patients without birch pollen allergy. Immunoblot inhibition experiments were used to investigate the immunological properties of natural LTP compared to the recombinant LTP. Potential cross-reactivity of natural LTP to pollens was studied by immunoblot and EAST inhibition tests. RESULTS: The LTP was identified as a 345 bp open reading frame coding for 92 amino acids of the mature protein and a signal peptide of 23 amino acids. Eight out of 16 Spanish sera displayed IgE reactivity to the recombinant hazelnut LTP, only one was positive to the birch-related rCor a 1.0401, and none to profilin. In contrast, none of the German patients with pollen-independent hazelnut allergy displayed IgE-reactivity to the recombinant LTP. The LTP was denominated as Cor a 8 and submitted to the allergen data base of the WHO/IUIS Allergen Nomenclature Subcommittee. IgE immunoblot inhibition experiments with hazelnut extract showed that natural Cor a 8 and recombinant Cor a 8 shared identical epitopes. EAST inhibition tests showed no significant cross-reactivity to birch and grass pollens but to plane, pellitory and mugwort pollen. In contrast, immunoblot analysis revealed no cross-reactivity against these pollens applying 100 µg pollen extract/cm gel. CONCLUSIONS: The lipid transfer protein is a major allergen for patients from Spain with hazelnut allergy accounting for potentially severe allergic reactions. In contrast, sensitisation to Cor a 8 seems to be irrelevant to patients in Germany suffering from hazelnut allergy. As shown by EAST inhibition tests, we hypothesise that cross-reactivity to Cor a 8 may be caused via the respiratory route by pollens, which are mainly grown in the Mediterranean area such as plane>mugwort>pellitory.

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[27] - Scheurer S, Haase T, Schocker F, Cisteró-Bahíma A, Enrique E, Becker WM, et al. Prevalence of IgE-Sensitization and Cross-Reactivity of Pru av 3, the Lipid Transfer Protein From Cherry. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°940

In Central and Northern Europe, food allergy to Rosaceae fruits is highly associated with birch pollinosis, owing to the existence of IgE cross-reactive homologous allergens in birch pollen and food. By contrast, in the Mediterranean area, severe allergic reactions to these fruits frequently are not related to birch pollen allergy, predominantly elicted by lipid transfer proteins (LTP). Recently, Pru av 3, the LTP from cherry has been identified as major allergen in Italian cherry-allergic subjects. The aim of this study was to evaluate the sensitization to Pru av 3 in a group of cherry allergic patients recruitated in Spain and to investigate the IgE-cross-reactivity of Pru av 3 with hazelnut LTP (Cor a 8) and with pollen extracts. All cherry-allergic patients (n=8) were allergic to plane tree pollen (Platanus acerifolia), and 4 out of all subjects investigated were allergic to grass pollen. The prevalence of IgE sensitization was investigated by EAST: Six out of 8 cherry-allergic patients were sensitized to rPru av 3, none was positive to the cherry profilin Pru av 4. Only one patient was sensitized to birch-related Pru av 1, identified as major allergen in the middle- and north European population. Two out of 8 cherry-allergic patients reported systemic reactions (angioedema) after ingestion of the corresponding food. IgE-Immunoblot and/or IgE-EAST (Enzyme Allergosorbent Test) inhibition experiments indicated a partial cross-reactivity between rPru av 3 and extracts from plane tree, ragweed, mugwort, and Parietaria pollen. No Inhibition of IgE-binding was found with the recombinant (r) hazelnut LTP Cor a 8, birch pollen and olive pollen extract as inhibitors. By contrast, IgE binding to recombinant hazelnut LTP was almost completely inhibited by rPru av 3. Preincubation of patient sera with cherry extract showed that natural and recombinant Pru av 3 shared identical IgE-epitopes. LTPs are potentially servere food allergens. The IgE cross-reactivity between food LTPs and pollen may indicate the presence of homologus pollen LTPs and their potential relevance in triggering food allergies by a primary sensitization by the inhalative route. Moreover, epitope similarities and differences of LTP-epitopes have to be taken into consideration in studies of cross-reactions in patients with multiple food allergies

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[28] - Lauer I, San Miguel-Moncín M, Hartz C, Cistero-Bahima A, Conti A, Vieths S, et al. The lipid transfer protein (LTP) from Platanus acerifolia pollen, Pla a 3, is a major allergen and cross-reacts with food LTPs. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1001

Background: Plane trees such as Platanus acerifolia are an important source of airborne allergens in many cities of Europe. Until now, only two major allergens of plane pollen, Pla a 1 and Pla a 2, have been characterised. In the Mediterranean area food-allergic patients are frequently sensitised to non-specific LTPs (nsLTPs). Sensitisation to food LTPs is not related to birch pollen allergy, and patients suffer from more severe reactions, in addition to OAS. An association between plane tree pollen allergy and plant food allergy was described, but the cross-reacting allergens have not been identified. The aim of this study was identification and characterisation of the nsLTP from plane pollen, and to investigate its immunological relationship to food LTPs from lettuce, cherry and hazelnut. Methods: Plane pollen extract was fractionated by ion exchange chromatography. Plane pollen LTP was identified by N-terminal amino acid sequence analysis. The prevalence of IgE reactivity to plane LTP was determined by immunoblotting using 45 Spanish plane pollen allergic patients with various plant food allergies and 14 plane pollen sensitised patients from Germany. In addition, sensitisation to LTPs from nuts, fruits and vegetables and their cross-reactivity to plane LTP was evaluated by immunoblotting and IgE inhibition experiments. Results: A 10 kDa IgE-reactive protein from plane pollen was partially purified and identified by N-terminal amino acid sequencing as nsLTP (Pla a 3). The N-terminal sequence of Pla a 3 showed the highest aa-ID of 58% with that of Lac s 1 (lettuce LTP). Pla a 3 was cross-reactive with food LTPs. Pla a 3 showed a prevalence of IgE reactivity of 66.6% (30/45) in Spanish plane pollen allergic patients. In contrast, Pla a 3 sensitisation was not relevant in plane pollen sensitised patients from Germany. We observed only partial correlation between sensitisation to plane pollen LTP and LTPs from lettuce, cherry and hazelnut. On the one hand, there were Spanish patients sensitised to food LTPs without plane pollen allergy, on the other hand, food LTP allergic patients without sensitisation to Pla a 3 were found. Conclusions: We have characterised the plane LTP as major allergen in plane pollen allergic patients with concomitant allergy to plant-derived foods recruited in the Mediterranean area. In Spanish patients sensitisation to food LTPs is not associated exclusively to the plane pollen LTP.

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[29] - Tordesillas L, Sanchez-Monge R, Cuesta-Herranz J, Compes E, Garcia-Carrasco B, Lombardero M, et al. A search for peach Pru P 3-homologous lipid transfer proteins in pollens. Allergy 2008;63(suppl. 88):117-118

Background: Two different patterns of sensitisation to Rosaceae fruits have been described in the Centre/North (Bet v 1-like as main allergens) and South (Lipid Transfer Proteins, LTPs, as major allergens) of Europe. Sensitisation to distinctive pollens have been claimed as a putative source of these differential profiles. Objetive: To explore the presence of putative Pru p 3-like allergens in Ambrosia artemissifolia (ragweed), Artemisia vulgaris (mugwort), Betula verrucosa (birch), Dactylis glomerata (barnyard grass), Helianthus annus (sunflower), Parietaria judaica (pellitory), Phleum pratensis (timothy), Plantago lanceolata (plantain) and Platanus occidentalis (plane tree) pollens. Methods: PBS extracts from the different pollens were separated by SDS-PAGE and immunodetected with anti-Pru p 3 polyclonal antibodies or IgE of sera from peach allergic patients (with associated pollinosis). ELISA-inhibition assays using purified Pru p3 and mugwort Art v 3 allergens as inhibitors were also performed. Pollen RNA samples and a cDNA encoding Pru p 3 probe were used in Northern analysis. Results: Pru p 3-like proteins were detected only in mugwort, but not in the other pollens analyzed, using either anti-Pru p 3 antibodies or sera from allergic patients. The cDNA-Pru p 3 probe hybridized with specific RNA bands from mugwort and plane tree pollens, but no band was found in the rest of pollens. Conclusion: Pru p 3-like LTPs were present in mugwort and plane tree pollens, as previously reported, but were not detected in ragweed, mugwort, birch, barnyard grass, sunflower, pellitory, timothy, plantain and plane tree pollens. A limited structural similarity with Pru p 3 and or even null levels of putative homologous allergens could explain the lack of detection of Pru p 3-like LTPs in most pollen analyzed.

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[30] - 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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[31] - Mittermann I, Fetrow JS, Schaak DL, Almo SC, Kraft D, Heberle-Bors E, et al. Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself ? Sex Plant Reprod 1998;11:183-191

Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. Profilins from birch pollen (Betula verrucosa (pendula)), humans and yeast (Schizosaccharomyces pombe) were expressed and purified from Escherichia coli and it was demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulfide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using 125I-labelled birch pollen, yeast and human profilin in overlay experiments, it was shown that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen. 56 ref.

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[32] - Metz-Favre C, Pauli G, Castro L, Valenta R, de Blay F. Demonstration of Bet v 2 responsibility in birch-induced symptoms. Allergy 2009;64(Suppl. 90):508

Background: Clinical relevance of profilin in respiratory symptoms has not yet been demonstrated. Bet v 1 is recognized as the major allergen in birch pollinosis in France. Case report: A 54-year-old man, born in Maurice island, had suffered from grass pollinosis for 13 years and had been desensitised with grass pollen for 4 years. He also complained of oral symptoms when eating melon, longan and avocado. Recently he complained of rhinitis in April (birch season). Skin prick tests were positive with grass, birch, ash, artemisia and plantago pollens as well as with melon, longan and avocado. Specific IgE serum measurements were performed only with recombinant allergens : they were significantly high to grass pollen molecular allergens (anti-rPhl p 1= 9,6 kU/l; anti-rPhl p 5 = 17.3 kU/l, anti-rPhl p 2= 4.3 kU/L, anti-rPhlp 12=1.6 kU/l). For birch molecular allergens, specific IgE serum levels were negative to Bet v 1, 4, and 6 but positive to Bet v 2 (1.4 kU/l). For ash pollen molecular allergens specific IgE against nOle e 1 were found at a low level of 0.21 kU/l. The nasal provocation test with birch extract (Allerbio-ALK, France) was positive in a dose dependent manner. In order to verify that grass profilin is the inducer of profilin sensitization we performed ELISA inhibition studies with the patient‚s serum. The inhibition experiment confirmed that the primary sensitizer was Phl p 12 because it was able to inhibit the IgE binding to Bet v 2 better than Bet v 2 did with Phl p 12. Conclusion: Sensitization to major allergens (Phl p 1,2, and 5) from grass pollen confirms grass pollinosis. Food allergy may be related to profilins. Sensitization to grass pollen and food profilins probably induced sensitization to birch profilin, as well as the subsequent symptoms during birch pollen season and the positive nasal challenge with birch pollen extract.

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[33] - van Ree R, Van Leeuwen WA, Akkerdaas JH, Aalberse RC. How far can we simplify in vitro diagnostics for Fagales tree pollen allergy ? A study with three whole pollen extracts and purified natural and recombinant allergens. Clin Exp Allergy 1999;29:848-855

BACKGROUND: Current diagnostic tests for Fagales tree pollen allergy are often composed of mixtures of pollen of birch, alder and hazel. Their complex composition hampers accurate standardization . OBJECTIVE: The aim of this study was to investigate whether mixtures of tree pollen extracts can be replaced by a single pollen species, and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens . METHODS: Sera (n = 1725) were selected on ground of a general suspicion for inhalant allergy, and tested in a RAST for birch, alder and hazel pollen. Sera with > 0.5 RU/mL for any of the three species were tested in a RAST for natural Bet v 1 and Bet v 2 as well as for recombinant versions of both allergens . RESULTS: Specific IgE antibodies (> 0.3 RU/mL) against birch, alder and hazel were found in 242, 298 and 292 sera, respectively. All sera with a positive RAST for alder and/or hazel and a negative RAST for birch were low-responder sera on alder and hazel, only five sera having a RAST value > 1.0 (all < 2.0). For all sera with a RAST > 0.5 RU/mL (n = 250), the mean of individual ratio's alder/birch and hazel/birch was 1.02 and 0.54, respectively. Of 223 of these sera, 63.2% had specific IgE against natural Bet v 1 and 63.7% against natural Bet v 2. When responses to both allergens were combined 93.7% were positive. The mean ratios Bet v 1 + 2/extract were 1.00, 1.04 and 2. 11 in case of birch, alder and hazel, respectively. For 211 sera the same analysis was performed with recombinant Bet v 1 and Bet v 2. Only six sera with Bet v 1-specific IgE (all < 0.5 RU/mL) were negative (< 0.3 RU/mL) on recombinant Bet v 1. For Bet v 2, 77/132 sera with specific IgE to the natural allergen did not react to the recombinant version. Twelve false-negatives had RAST values > 1.0 RU/mL. The mean of the individual recombinant/natural ratios was 0. 98 for Bet v 1 and 0.38 for Bet v 2 (P < 0.001). The mean ratio rBet v 1 + 2/birch was 0.75 with 17.5% false-negatives on the combination of recombinant allergens . CONCLUSION: Reliable in vitro diagnosis is possible with a single tree pollen extract (birch or alder). The same is true for purified natural Bet v 1 and Bet v 2. A combination of recombinant molecules is slightly less efficient.

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[34] - 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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[35] - Scala E, Zaffiro A, Quaratino D, D'Ambrosio C, Bernardi M, Palazzo P, et al. IgE Recognition Pattern of Homologous Allergens Tested by Microarray-based Nanotechnology. J Allergy Clin Immunol 2007;119(1 suppl):S105

RATIONALE: IgE recognition of homologous molecules shared by inhalant and food allergen sources are often seen in clinical practice. The aim of our study was to evaluate through allergen microarray technique the IgE recognition pattern of the allergic population. METHODS: 1110 individuals (42/58 male/female) with a clinical history of urticaria, rhinitis, asthma and atopic dermatitis have been recruited and tested by allergen-based microarray system (VBC-Genomic, Vienna, Austria). Patients were grouped on the basis of their reactivity to at least one allergen in one of the following three groups: Profilins (Bet v 2, Cyn d 12, Hel a 2, Hev b 8, Mer a 1, Ole e 2, Par j 3, Phl p 12, Pho d 2); Bet v 1-related allergens (Aln g 1, Api g 1, Bet v 1a, Bet v 1d, Cor a 1, Dau c 1 and Mal d 1.0108); tropomyosins (Ani s 3, Der p 10, Hel as 1, Pen i 1, Pen m 1, Per a 7). RESULTS: 523 (47%) subjects were positive to at least one Bet v 1-related allergens, 415 (37%) to at least one Profilin, 123 (11.1%) to at least one tropomyosin. Bet v 1 (85%) was the most frequently IgE positive molecule within Bet v 1-related allergen reactive individuals, whereas Mer a 1 (89%) ranked first within the profilin subset, and Der p 10 (80%) was the most IgE recognized tropomyosin. Stratifying subjects by IgE concentrations showed an almost homogeneous recognition pattern for all profilins and tropomyosins, whereas IgE to some Bet v 1-like molecules from foods were not detected even at high Bet v 1-IgE concentration. CONCLUSIONS: Panel of grouped allergens to be tested by means of microarray system are helpful to fully describe homologous allergenic molecule immunological relationship. IgE reactivity pattern could relate to patient clinical picture.

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[36] - Seiberler S, Scheiner O, Kraft D, Lonsdale D, Valenta R. Characterization of a birch pollen allergen, Bet v III, representing a novel class of Ca2+ binding proteins: specific expression in mature pollen and dependence of patients' IgE binding on protein-bound Ca2+. EMBO J 1994;13:3481-3486

A cDNA coding for a birch pollen allergen, Bet v III, with significant sequence homology to Ca2+ binding proteins was isolated from an expression cDNA library using serum IgE from a patient who was allergic to pollen. The deduced amino acid sequence of the pollen allergen contained three typical Ca2+ binding sites. Peptides mimicking the Ca2+ binding sites of Bet v III were synthesized and shown to bind 45Ca in blot overlays. The binding of patients' IgE to the recombinant allergen depended on the native protein conformation and protein-bound Ca2+. Depletion of Ca2+ led to a reversible loss of the IgE binding thus representing a conformational IgE epitope adopted by a polypeptide upon Ca2+ binding. By RNA hybridization it was demonstrated that Bet v III is expressed preferentially in mature pollen. Bet v III therefore represents a pollen allergen which because of its unique structural features also belongs to a novel class of Ca2+ binding proteins.

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[37] - Neudecker P, Nerkamp J, Eisenmann A, Nourse A, Lauber T, Schweimer K, et al. Solution structure, dynamics, and hydrodynamics of the calcium-bound cross-reactive birch pollen allergen Bet v 4 reveal a canonical monomeric two EF-hand assembly with a regulatory function. J Mol Biol 2004;336:1141-1157

Birch pollinosis is one of the prevailing allergic diseases. In all, 5-20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca2+-binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal alpha-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca2+ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.

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[38] - Mittermann I, Fetrow JS, Schaak DL, Almo SC, Kraft D, Heberle-Bors E, et al. Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself ? Sex Plant Reprod 1998;11:183-191

Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. Profilins from birch pollen (Betula verrucosa (pendula)), humans and yeast (Schizosaccharomyces pombe) were expressed and purified from Escherichia coli and it was demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulfide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using 125I-labelled birch pollen, yeast and human profilin in overlay experiments, it was shown that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen. 56 ref.

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[39] - Ferreira F, Engel E, Briza P, Richter K, Ebner C, Breitenbach M. Characterization of recombinant Bet v 4, a birch pollen allergen with two EF-hand calcium-binding domains. Int Arch Allergy Immunol 1999;118:304-305

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[40] - Engel E, Richter K, Obermeyer G, Briza P, Kungl AJ, Simon B, et al. Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains. J Biol Chem 1997;272:28630-28637

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.

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[41] - Karamloo F, Schmitz N, Scheurer S, Foetisch K, Hoffmann A, Haustein D, et al. Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins. J Allergy Clin Immunol 1999;104:991-999

Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.

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[42] - 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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[43] - Wellhausen A, Schöning B, Petersen A, Vieths S. IgE binding to a new cross-reactive structure: a 35 kDa protein in birch pollen, exotic fruit and other plant foods. Z Ernahrungswiss 1996;35:348-355

Food allergies in birch pollen allergic patients have been shown to be due to cross-reactivities of specific IgE antibodies which are directed against birch pollen allergens with related proteins in fruit, nuts and vegetables. We identified a new cross-reactive structure of 35 kDa in birch pollen and some plant food extracts by Enzyme Allergosorbent Test (EAST) and immunoblot inhibition studies. The 35 kDa birch pollen protein is a minor allergen to which approximately 10-15% of birch pollen allergic individuals have specific IgE. Our data demonstrate that there is cross-reactivity of this protein with proteins of comparable size from lychee, mango, banana, orange, apple, pear and carrot. While the 35 kDa protein is immunologically independent of the major birch pollen allergen Bet v 1, we also observed IgE binding to a 34 kDa structure which appears to be a Bet v 1 dimer.

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[44] - Wellhausen A, Schöning B, Petersen A, Vieths S. IgE binding to a new cross-reactive structure: a 35 kDa protein in birch pollen, exotic fruit and other plant foods. Z Ernahrungswiss 1996;35:348-355

Food allergies in birch pollen allergic patients have been shown to be due to cross-reactivities of specific IgE antibodies which are directed against birch pollen allergens with related proteins in fruit, nuts and vegetables. We identified a new cross-reactive structure of 35 kDa in birch pollen and some plant food extracts by Enzyme Allergosorbent Test (EAST) and immunoblot inhibition studies. The 35 kDa birch pollen protein is a minor allergen to which approximately 10-15% of birch pollen allergic individuals have specific IgE. Our data demonstrate that there is cross-reactivity of this protein with proteins of comparable size from lychee, mango, banana, orange, apple, pear and carrot. While the 35 kDa protein is immunologically independent of the major birch pollen allergen Bet v 1, we also observed IgE binding to a 34 kDa structure which appears to be a Bet v 1 dimer.

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[45] - Bolhaar STHP, van Ree R, Bruijnzeel-Koomen CAFM, Vieths S, Hoffmann-Sommergruber K, Knulst AC, et al. Severe Allergy to Sharon Fruit Caused by Birch Pollen. Int Arch Allergy Immunol 2005;136:45-52

BACKGROUND: Allergy to sharon fruit (persimmon) has been only rarely reported. Cross-reactivity with pollen (profilin and Bet v 6) appeared to be involved, but Bet v 1 has not been implicated previously . OBJECTIVE: It is our aim to identify whether Bet v 1 sensitization is linked to sharon fruit allergy . METHODS: Two patients with a reaction upon first exposure to sharon fruit were included in the study, as well as 7 patients with birch-pollen-related apple allergy. Sensitivity was assessed by skin prick testing (SPT), a radio-allergosorbent test (RAST) and immunoblotting. RAST analysis was performed for Bet v 1, Bet v 2 and Bet v 6. Cross-reactivity was evaluated by RAST and immunoblot inhibitions. Biological activity of IgE was measured by basophil histamine release. Sharon fruit allergy was evaluated by double-blind placebo-controlled food challenge (DBPCFC) or open challenge (OC) . RESULTS: Both sharon-fruit-allergic patients demonstrated positive reactions in the RAST (8.6 and 6.2 IU/ml, respectively) and SPT (wheal area 37 and 36 mm2). Sharon fruit allergy was confirmed by DBPCFC in 1 patient. The second patient refused a challenge because of the severe initial reaction. Sera from both patients were reactive to Bet v 1 and Bet v 6, which was cross-reactive with sharon fruit by inhibition assays. The patient with the severest reactions was reactive to profilin on immunoblotting. However, profilin did not induce significant histamine release, nor did Bet v 6. Bet v 1 induce approximately 60% histamine release. An OC with sharon fruit in 7 patients allergic to birch pollen and apple, who had not eaten sharon fruit previously, was positive in 6/7 cases . CONCLUSIONS: Birch-pollen-related allergy to sharon fruit is mediated by the known cross-reactive pollen allergens including Bet v 1 and may become more of a problem should sharon fruit consumption increase.

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[46] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.

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[47] - Vieths S, Frank E, Scheurer S, Meyer HE, Hrazdina G, Haustein D. Characterization of a new IgE-binding 35-kDa protein from birch pollen with cross-reacting homologues in various plant foods. Scand J Immunol 1998;47:263-272

The present investigation was undertaken to obtain molecular data of a new immunoglobulin (Ig)E-binding birch pollen protein with a mass of 35 kDa. In a previous study, this protein showed IgE cross-reactivity with 34- and 35-kDa proteins in apples, pears, carrots, bananas and other exotic fruits. Since the protein was N-terminally blocked, it was purified by preparative SDS-PAGE, and multiple proteolytic fragments were subsequently generated by in-gel digestion with the endoproteinases Glu C, Lys C and Clostripain. After electrophoretic separation and blotting onto polyvinylidene difluoride (PVDF), the resulting polypeptides were subjected to N-terminal amino acid microsequencing. The internal sequences obtained showed a high degree of sequence identity to isoflavone reductases (IFR) and isoflavone reductase-like proteins (IRL) from several plants which also had a similar size. For a stretch of 25 consecutive residues this identity ranged from 56% for IFR from peas and chick peas and an IRL from maize, to 80% for a tobacco IRL. A 453 bp fragment was amplified from total birch pollen RNA by polymerase chain reaction (PCR) using primers derived from the nucleotide sequence of the tobacco IRL. The deduced 151 amino acid sequence represented approximately 50% of the protein and confirmed the sequence identities obtained by Edman degradation. Moreover, the 25 amino acid sequence was included in the cloned fragment. Deduced and determined amino acids showed only one mismatch, which was due to a single nucleotide exchange. At the antibody level, the immunological relationship of the birch pollen protein to IRL and IFR was demonstrated by immunoblotting with a rabbit antiserum against a pea IFR which recognized the same birch protein as patients' IgE. The rabbit antiserum also reproduced the cross-reactivity pattern previously observed with patients' IgE by recognizing related proteins in specific plant foods, including some exotic fruits. We therefore suggest that the 35-kDa birch pollen protein belongs to the IFR/IRL family and represents a minor allergen, possibly being responsible for less common pollen-related food allergies in patients allergic to birch pollen.

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[48] - Karamloo F, Schmitz N, Scheurer S, Foetisch K, Hoffmann A, Haustein D, et al. Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins. J Allergy Clin Immunol 1999;104:991-999

Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.

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[49] - Cadot P, Diaz JF, Proost P, Van Damme J, Engelborghs Y, Stevens EAM, et al. Purification and characterization of an 18-kD allergen of birch (Betula verrucosa) pollen: identification as a cyclophilin. J Allergy Clin Immunol 2000;105:286-291

Five birch pollen allergens have been identified so far. In a previous study we detected new birch pollen allergens with an isoelectric point in the range 9.0 to 9.3, present only in extracts prepared at controlled basic pH. OBJECTIVE: The purpose of the current study was to purify and characterize those allergens. METHODS: The target allergens were purified by ion exchange and hydrophobic interaction chromatography. Analyses were carried out by SDS-PAGE, isoelectric focusing, immunoblotting, and amino acid sequencing. The in vivo reactivity of the allergens was evaluated by skin testing. RESULTS: An 18-kd protein, which we named Bet v 7, was purified. This 18-kd protein corresponded to 3 bands on isoelectric-focusing immunoblots that probably represent isoforms. On immunoblots up to 20.8% of birch pollen-allergic patients recognized those allergens. The clinical relevance of Bet v 7 was demonstrated by positive immediate-type skin testing on a patient allergic to birch pollen. Sequencing of an internal peptide yielded an amino acid sequence showing high homology with various plant cyclophilins. The rotamase activity of the protein, inhibited by cyclosporin A, further confirmed that Bet v 7 belongs to the group of cyclophilins. CONCLUSION: We have purified a novel allergen of birch pollen, Bet v 7, belonging to the cyclophilin family. Because cyclophilins are highly conserved proteins over the phylogeny, we may postulate that Bet v 7 is a member of a new family of panallergens.

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[50] - Fujita C, Moriyama T, Ogawa T. Identification of cyclophilin as an IgE-binding protein from carrots. Int Arch Allergy Immunol 2001;125:44-50

BACKGROUND: Plant food allergies have been associated with pollenosis, although most of the causative allergens are as yet undefined. It is important to elucidate the properties of plant food allergens in order to minimize a patient's risks in food selection. The purpose of the present study was to examine and characterize the IgE-binding proteins in carrots as possible allergens by using patients' sera. METHOD: IgE-binding proteins in carrot extract were screened by an immunoblot technique using sera of patients with atopic dermatitis (selected based upon a case history of food allergies). An allergenic protein was purified from carrot extract by chromatographic procedures. The N-terminal amino acid sequence of allergenic protein was determined and subjected to a computer homology search. Cross-reactivity between carrot and birch allergens was examined by immunoblotting. RESULTS AND CONCLUSION: A unique, approximately 20-kDa allergenic protein that reacted with about 14% of patients' sera was isolated and characterized. The N-terminal amino acid sequence of the purified protein was found to be homologous with those of plant cyclophilins. This allergen exhibited a peptidyl-prolyl cistrans isomerase activity, which was inhibited by the conjugation of cyclosporin A. These properties of the allergenic protein isolated from carrot identified it as a cyclophilin, a possible plant food allergen. No cross-reactivity between this 20-kDa carrot allergen and Bet v 7, a birch pollen cylcophilin, was observed.

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[51] - Cadot P, Nelles L, Srahna M, Dilissen E, Huyle broeck D, Ceuppens JL. Cloning and expression of Bet v 7, the birch cyclophilin. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster

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[52] - Cadot P, Nelles L, Srahna M, Dilissen E, Ceuppens JL. Cloning and expression of the cyclophilin Bet v 7, and analysis of immunological cross-reactivity among the cyclophilin A family. Mol Immunol 2006;43:226-235

Allergic symptoms in sensitized individuals are caused by proteins named allergens. We report here the cloning and the production of the cyclophilin Bet v 7, one of the birch pollen allergens. Recombinant Bet v 7 was produced in bacteria and used to raise a rabbit anti-Bet v 7 antiserum. With this antiserum we detected cyclophilin A in several pollen species and we demonstrated immunological cross-reactivity among those plant cyclophilins A by immunoblot and ELISA inhibition experiments. However, we could not detect cyclophilins in extracts of animal or mould origin with our anti-Bet v 7 antiserum. By inhibition experiments with purified mould cyclophilins, we confirmed the absence of cross-reactivity between plant cyclophilins and non-plant cyclophilins. In addition, our results indicate that the level of immunological cross-reactivity correlates with the level of sequence identity among the cyclophilin A family. This allowed us to define the plant cyclophilin A sub-family as being immunologically distinct, which might have implications at the clinical level in the allergy practice.

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[53] - Mahler V, Fischer S, Heiss S, Duchêne M, Kraft D, Valenta R. cDna cloning and characterization of a cross-reactive birch pollen allergen: identification as a pectin esterase. Int Arch Allergy Immunol 2001;124:64-66

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[54] - Stumvoll S, Lidholm J, Thunberg R, DeWitt AM, Eibensteiner P, Swoboda I, et al. Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential. Biol Chem 2002;383:1383-1396

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

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[55] - Leduc-Brodard V, Inacio F, Jaquinod M, Forest E, David B, Peltre G. Characterization of Dac g 4, a major basic allergen from Dactylis glomerata pollen. J Allergy Clin Immunol 1996;98:1065-1072

Monoclonal antibodies were produced against Dac g 4, a purified major basic allergen from Dactylis glomerata pollen. Their ability to be used for immunopurification of Dac g 4 was studied on a BIAcore apparatus (Pharmacia). The allergen was purified by affinity chromatography with one monoclonal antibody. Its precise molecular mass, 59,185 +/- 30 d, was determined by mass spectrometry. Its isoelectric point is 10.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting showed that Dac g 4-related proteins of similar molecular mass were detected in the majority of allergenic grass pollen species. By double-site ELISAs, we have estimated that Dac g 4 represents about 6% of the total proteins from a water-soluble extract. One monoclonal antibody (mAb H) recognized a 60 kd cross-reactive protein in other grass pollens, though none in any of the tree or weed pollens tested. Inhibition studies of IgE antibody binding to Dac g 4 with pollen extracts confirmed the presence of cross-reactive allergens in Secale cereale, Lolium perenne, Festuca elatior, Holcus lanatus, Bromus arvensis, Poa pratense, Hordeum sativum, and Phleum pratense.

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[56] - 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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[57] - Grozdanovic M, Burazer L, Vuckovic O, Cirkovic-Velickovic T, Jankov R, Gavrovic-Jankulovic M. Characterisation of a thaumatin-like homologue from birch (Betula verrucosa) pollen. Allergy 2009;64(Suppl. 90):249

Background: Thaumatin-like proteins have been established as an allergen family present in pollen and plant foods. Allergenic TLPs have been described in pollen grains of the mountain cedar (Jun a 3; Juniperus ashei), in Cupresus arizonica pollen (Cup a 3), and in Japanese cedar (Cry j 3; Cryptomeria japonica). They are also found in apple, cherry, bell pepper, grape and orange fruit. The aim of this study was to explore the presence and IgE reactivity of a TLP homologue in birch pollen. Methods: Birch pollen (Betula verrucosa) protein extract was fractionated on an ion-exchange chromatography column, and the protein of interest was further purified by means of reverse-phase chromatography. Protein homogeneity was analysed by SDS-PAGE and 2D PAGE. A 2D immunoblot of the purified protein was performed using monoclonal anti-TLP antibodies. Allergenicity of the purified protein was examined by immunoblot with sera of eight birch allergic patients, with specific IgE above CAP class 3. Results: A purified protein from birch pollen revealed a molecular mass of about 25 kDa in reducing SDS PAGE and an acidic pI. Purified protein was bound by anti-TLP monoclonal antibodies. In Western blot analysis four out of eight patients showed IgE reactivity to the purified protein, asserting the allergenic properties of this TLP homologue. Conclusion:We have partially characterized a thaumatin-like homologue present in Betula verrucosa pollen. Further structural characterization, as well as immunological studies performed on a larger scale, will determine its clinical significance and eventual contribution to birch pollen-fruit syndrom.

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[58] - Yamagiwa M, Hattori R, Ito Y, Yamamoto S, Kanba M, Tasaki T, et al. Birch-pollen sensitization in an area without atmospheric birch pollens. Auris Nasus Larynx 2002;29:261-266

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[59] - Diez-Gomez ML, Quirce S, Cuevas M, Sanchez-Fernandez C, Baz G, Moradiellos FJ, et al. Fruit-pollen-latex cross-reactivity: implication of profilin (Bet v 2). Allergy 1999;54:951-961

An association between allergy to fruits and latex, and between pollen and plant-derived food has been described. The cross-reactive structures responsible for these associations have not yet been completely elucidated. METHODS: IgE reactivity to the recombinant allergens Bet v 1 and Bet v 2, different pollens, natural latex, papain, and bromelain was investigated in 29 patients with allergy to fruits or vegetables who lived in an area without birch trees. RESULTS: Exactly 79.3% of patients were allergic to grass pollen, and two of them had clinical allergy to latex. Serum IgE reactivity (CAP) to birch pollen was found in 65% of patients, to Bet v 2 in 51.7%, to Bet v 1 in 3.4%, to latex in 58.6%, to bromelain in 51.7%, and to papain in 17.2% of patients. All subjects with positive IgE to Bet v 2 had also reactivity to latex, grass, olive tree, birch, and mugwort pollens. The six patients not allergic to pollen did not show IgE reactivity to latex, Bet v 1, or Bet v 2. A significant correlation was found between CAP to latex with Bet v 2 (r=0.86, P<0.001), with birch (r=0.86, P<0.001), and with ryegrass (r=0.81, P<0.001). Immunoblotting using nine sera with positive CAP to birch pollen showed IgE-binding to a 15-kDa band that was recognized by antiprofilin monoclonal antibody. Bet v 2 CAP could be inhibited up to 52% by ryegrass and up to 23% by mugwort. CAP to latex was almost completely inhibited by ryegrass pollen with sera from five subjects without symptoms due to latex, whereas no inhibition was observed with serum from one patient with allergy to latex. CONCLUSIONS: Patients with allergy to plant-derived food and associated pollinosis showed a high frequency of IgE reactivity to Bet v 2, which may cause positive serum IgE determinations to latex and birch pollen due to the presence of cross-reactive epitopes. IgE reactivity to Bet v 2 may serve as an indicator of broad sensitization.

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[60] - Huertas AJ, Lavín JR, García-Cervantes AM. Sensitization to birch pollen in a Mediterranean area without Betulaceae family plants. Allergy Clin Immunol Int 2005;17(Suppl. 1):222

Birch pollen is a relevant allergen in some countries of northern and central Europe, where this tree is abundant. Also they have been found sensitizations to this pollen in other areas without birch tree. The aim of this study is to determine the incidence of the skin sensitization to birch pollen in a group of patients with pollinosis who live in Cartagena (Spain), a border Mediterranean sea city, where Betulaceae family plants are not present. Material and Methods: We selected 183 pollen-allergic patients, 102 male and 81 women included of consecutive way, whit symptoms of seasonal rhinitis and/or asthma. Prick test were performed with a battery of inhalant allergens that included the principal allergenic pollen: Betula alba, Olea europaea, grasses-mix, Cynodon dactylon, Plantago lanceolata, Platanus spp, Artemisia vulgaris, Salsola kali, Parietaria judaica, Chenopodium album and Cupressus arizonica. The patients were distributed in three groups depend on the number of sensitized pollen: A (1 to 3), B (4 to 6) and C (more than 6). Results: The total sample included 183 patients, with a mean age of 28,61 years (range 4-76). The distribution among groups was: A:114 patients (62,30%); B: 43 (23,50%) and C: 26 (14,20%). We found 30 patients (16,39%) sensitized to birch pollen, distributed on the following way: group A: 1 (3,33%); group B: 8 (26,67%) and group C: 21 (70%).We did not find monosensitized to this pollen. Distribution was different in no-sensitized patients: A: 110 (71,90%); B: 36 (23,53%); C: 7 (4,57%). Conclusions: 1. -The sensitization to birch pollen is common in pollen-allergic patients in our area. 2. -These results suggest that the found birch pollen sensitization would be the expression of a vegetable panallergen sensitization, probably a profilin.

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[61] - Mari A, Wallner M, Ferreira F. Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4). Clin Exp Allergy 2003;33:1419-1428

BACKGROUND: Birch allergy is one of the most common pollinosis in areas where exposure to high levels of birch pollen is common. Little is known about birch sensitivity in areas without birch pollen exposure and reactivity to birch-related species within the Fagales order . OBJECTIVE: the aim was to evaluate Fagales reactivity within a population not exposed to birch pollen using epidemiological, diagnostic, and laboratory approaches by means of extracts and allergenic molecules . METHODS: A cohort of 5335 respiratory allergic patients was screened by means of skin testing birch, hazel, and oak pollen extracts. Patients were from a birch-free area, but exposed to other Fagales pollen species. A subset of patients was from an intensively cultivated hazel area. A sample of the Fagales allergic population was tested with other Fagales pollen extract (alder, hornbeam, beech, chestnut) and with apple and hazelnut. IgE detection was performed with birch, hazel, oak, apple, and hazelnut extracts, and with Bet v 1, Bet v 2, Bet v 4, and bromelain. IgE immunoblots were performed using birch and hazel extracts. Epidemiological, clinical, and laboratory data were analysed by stratifying the allergic population . RESULTS: Twenty-five percent of the pollen allergic cohort was skin test positive to at least one of the three Fagales species. Combined reactivity to the three species was recorded in 80% of this cohort. Isolated hazel pollen reactivity was recorded in 13.5% of the Fagales allergic patients. Sixty-six percent of these subjects were from the intensively cultivated hazel area. Reactivity to apple and hazelnut was detected by skin test (40%) and IgE reactivity (60%), but only 19% of the positive patients reported symptoms related to at least one of the two foods. Reactivity to Bet v 1 was recorded in 84% of the birch/hazel/oak co-reactivity group, and in 28% of the subjects with the same co-reactivity, but associating a multiple pollen sensitization. IgE to Bet v 2 (50%) and Bet v 4 (23%) panallergens were recorded positive in the latter subset. Bet v 1 prevalence ranged between 48% and 21% among subgroups of patients coming from different areas. Furthermore, an IgE reactivity to hazel-restricted allergenic components was detected among subjects coming from the same area and having a hazel isolated reactivity . CONCLUSION: Fagales allergy can be found in birch-free areas caused by the exposure to other Fagales species. Birch allergens can be useful for mimicking the allergenic extract, but are also the exclusive tools for a fine diagnostic and epidemiological approach to Fagales pollen allergy. Allergenic molecules from the hazel family will increase the panel of available reagents for the molecule-based approach to allergy diagnosis and therapy.

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[62] - 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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[63] - Skypala I, Calderon M, Leeds A, Durham S. Pollen-food syndrome in United Kingdom subjects - which aeroallergens are commonly involved ? Allergy 2007;62(suppl. 83):358

Pollen food syndrome (PFS) is common in individuals with birch pollen sensitivity who present with hay fever symptoms in the spring. The aim of this study was to evaluate which pollen(s), apart from birch, are associated with PFS in UK-based subjects, and whether aero-allergen sensitivities differ in those subjects with springtime hay fever with/without associated PFS. Adult subjects reporting hay fever symptoms between March and May were included in the study. All subjects underwent skin prick testing with tree (birch, oak, beech, plane and three trees ˜ birch, alder and hazel), timothy grass, mugwort, latex, cat and house dust mite (D. pteronyssinus) allergens (ALK Abelló Denmark). A diagnosis of PFS was established for each subject using a combination of results from a standardised medical assessment, prick by prick testing with suspect foods and oral food challenge. 119 subjects completed the study; 92% were sensitised to one or more aeroallergens. Although all subjects reported spring and/or summer time hay fever, only 85% of the cohort were sensitised to pollen. Grass was the commonest sensitising pollen allergen (68%), followed by birch (64%), oak and three trees (53%), beech (34%), plane (26%) and mugwort (18%). Approximately half of the cohort were sensitised to cat and/or house dust mite, but less than 10% to latex. Subjects diagnosed with PFS had a significantly greater number of positive tests for birch, oak, beech (p<0.001) and grass (p<0.05) than those without PFS, but there was no significant difference between the groups for the other aeroallergens. Polysensitisation to pollen allergens occurred in 93% of the group diagnosed with PFS compared to 37% of the group without PFS. No aeroallergen sensitivities were individually or collectively significant predictors of the diagnosis of PFS although a combination of birch, grass and oak gave the best standardised beta coefficient. UK subjects with springtime hay fever are most likely to be sensitised to birch, grass and oak. The frequency of sensitivities to these allergens is greater in those who additionally have PFS. In contrast, subjects with/without PFS do not differ in their rate of sensitisation to cat and house dust mite. Sensitisation to pollen cannot predict who with springtime hay fever will have PFS, but those with PFS are more likely to be co-sensitised to several tree and grass pollens.

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[64] - Wensing M, Akkerdaas JH, van Leeuwen A, Stapel SO, Bruijnzeel-Koomen CAFM, Aalberse RC, et al. IgE to Bet v 1 and profilin: Crossreactivity patterns and clinical relevance. J Allergy Clin Immunol 2002;110:435-442

BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants . OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance . METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition . RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon . CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.

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[65] - van Ree R, Akkerdaas JH, Van Leeuwen A, Fernández-Rivas M, Asero R, Knul-Brettlova V, et al. New perspectives for the diagnosis of food allergy. Allergy Clin Immunol Int 2000;12:7-12

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[66] - Mari A, Wallner M, Ferreira F. Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4). Clin Exp Allergy 2003;33:1419-1428

BACKGROUND: Birch allergy is one of the most common pollinosis in areas where exposure to high levels of birch pollen is common. Little is known about birch sensitivity in areas without birch pollen exposure and reactivity to birch-related species within the Fagales order . OBJECTIVE: the aim was to evaluate Fagales reactivity within a population not exposed to birch pollen using epidemiological, diagnostic, and laboratory approaches by means of extracts and allergenic molecules . METHODS: A cohort of 5335 respiratory allergic patients was screened by means of skin testing birch, hazel, and oak pollen extracts. Patients were from a birch-free area, but exposed to other Fagales pollen species. A subset of patients was from an intensively cultivated hazel area. A sample of the Fagales allergic population was tested with other Fagales pollen extract (alder, hornbeam, beech, chestnut) and with apple and hazelnut. IgE detection was performed with birch, hazel, oak, apple, and hazelnut extracts, and with Bet v 1, Bet v 2, Bet v 4, and bromelain. IgE immunoblots were performed using birch and hazel extracts. Epidemiological, clinical, and laboratory data were analysed by stratifying the allergic population . RESULTS: Twenty-five percent of the pollen allergic cohort was skin test positive to at least one of the three Fagales species. Combined reactivity to the three species was recorded in 80% of this cohort. Isolated hazel pollen reactivity was recorded in 13.5% of the Fagales allergic patients. Sixty-six percent of these subjects were from the intensively cultivated hazel area. Reactivity to apple and hazelnut was detected by skin test (40%) and IgE reactivity (60%), but only 19% of the positive patients reported symptoms related to at least one of the two foods. Reactivity to Bet v 1 was recorded in 84% of the birch/hazel/oak co-reactivity group, and in 28% of the subjects with the same co-reactivity, but associating a multiple pollen sensitization. IgE to Bet v 2 (50%) and Bet v 4 (23%) panallergens were recorded positive in the latter subset. Bet v 1 prevalence ranged between 48% and 21% among subgroups of patients coming from different areas. Furthermore, an IgE reactivity to hazel-restricted allergenic components was detected among subjects coming from the same area and having a hazel isolated reactivity . CONCLUSION: Fagales allergy can be found in birch-free areas caused by the exposure to other Fagales species. Birch allergens can be useful for mimicking the allergenic extract, but are also the exclusive tools for a fine diagnostic and epidemiological approach to Fagales pollen allergy. Allergenic molecules from the hazel family will increase the panel of available reagents for the molecule-based approach to allergy diagnosis and therapy.

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[67] - Diez-Gomez ML, Quirce S, Cuevas M, Sanchez-Fernandez C, Baz G, Moradiellos FJ, et al. Fruit-pollen-latex cross-reactivity: implication of profilin (Bet v 2). Allergy 1999;54:951-961

An association between allergy to fruits and latex, and between pollen and plant-derived food has been described. The cross-reactive structures responsible for these associations have not yet been completely elucidated. METHODS: IgE reactivity to the recombinant allergens Bet v 1 and Bet v 2, different pollens, natural latex, papain, and bromelain was investigated in 29 patients with allergy to fruits or vegetables who lived in an area without birch trees. RESULTS: Exactly 79.3% of patients were allergic to grass pollen, and two of them had clinical allergy to latex. Serum IgE reactivity (CAP) to birch pollen was found in 65% of patients, to Bet v 2 in 51.7%, to Bet v 1 in 3.4%, to latex in 58.6%, to bromelain in 51.7%, and to papain in 17.2% of patients. All subjects with positive IgE to Bet v 2 had also reactivity to latex, grass, olive tree, birch, and mugwort pollens. The six patients not allergic to pollen did not show IgE reactivity to latex, Bet v 1, or Bet v 2. A significant correlation was found between CAP to latex with Bet v 2 (r=0.86, P<0.001), with birch (r=0.86, P<0.001), and with ryegrass (r=0.81, P<0.001). Immunoblotting using nine sera with positive CAP to birch pollen showed IgE-binding to a 15-kDa band that was recognized by antiprofilin monoclonal antibody. Bet v 2 CAP could be inhibited up to 52% by ryegrass and up to 23% by mugwort. CAP to latex was almost completely inhibited by ryegrass pollen with sera from five subjects without symptoms due to latex, whereas no inhibition was observed with serum from one patient with allergy to latex. CONCLUSIONS: Patients with allergy to plant-derived food and associated pollinosis showed a high frequency of IgE reactivity to Bet v 2, which may cause positive serum IgE determinations to latex and birch pollen due to the presence of cross-reactive epitopes. IgE reactivity to Bet v 2 may serve as an indicator of broad sensitization.

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[68] - Daschner A, Crespo JF, Pascual CY. Specific IgE to recombinant vegetal panallergen (rBet v 2) and fruit allergy in pollinic patients. Allergy 1999;53:614-618

The presence of IgE antibodies to the allergens rBet v 1 and rBet v 2 was investigated in patients with known sensitization to rye grass (Lolium perenne) and/or olive (Olea europaea) pollen, by comparing a group of 10 patients who had allergic symptoms after ingestion of fruits [including bananas, figs, kiwifruit, muskmelons, oranges and peaches] with a group of 17 patients who had only seasonal respiratory symptoms. There was no significant difference between the 2 groups for total IgE. All patients showed specific IgE to both L. perenne and O. europaea. No specific IgE binding to rBet v 1 was detected in any patient. The incidence of the presence of IgE antibodies to rBet v 2 was 90% in the group with fruit allergy and 35% in the group [without fruit allergy]. There was a significant association between presence of IgE antibodies to rBet v 2 and fruit allergy (P = 0.007). Specific IgE values to O. europaea pollen were higher in the fruit-allergy group than the group without fruit allergy (P = 0.032). It is concluded that pollen-allergic patients with specific IgE to birch pollen profilin show a significantly elevated frequency of fruit allergy.

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[69] - Diez-Gomez ML, Quirce S, Cuevas M, Sanchez-Fernandez C, Baz G, Moradiellos FJ, et al. Fruit-pollen-latex cross-reactivity: implication of profilin (Bet v 2). Allergy 1999;54:951-961

An association between allergy to fruits and latex, and between pollen and plant-derived food has been described. The cross-reactive structures responsible for these associations have not yet been completely elucidated. METHODS: IgE reactivity to the recombinant allergens Bet v 1 and Bet v 2, different pollens, natural latex, papain, and bromelain was investigated in 29 patients with allergy to fruits or vegetables who lived in an area without birch trees. RESULTS: Exactly 79.3% of patients were allergic to grass pollen, and two of them had clinical allergy to latex. Serum IgE reactivity (CAP) to birch pollen was found in 65% of patients, to Bet v 2 in 51.7%, to Bet v 1 in 3.4%, to latex in 58.6%, to bromelain in 51.7%, and to papain in 17.2% of patients. All subjects with positive IgE to Bet v 2 had also reactivity to latex, grass, olive tree, birch, and mugwort pollens. The six patients not allergic to pollen did not show IgE reactivity to latex, Bet v 1, or Bet v 2. A significant correlation was found between CAP to latex with Bet v 2 (r=0.86, P<0.001), with birch (r=0.86, P<0.001), and with ryegrass (r=0.81, P<0.001). Immunoblotting using nine sera with positive CAP to birch pollen showed IgE-binding to a 15-kDa band that was recognized by antiprofilin monoclonal antibody. Bet v 2 CAP could be inhibited up to 52% by ryegrass and up to 23% by mugwort. CAP to latex was almost completely inhibited by ryegrass pollen with sera from five subjects without symptoms due to latex, whereas no inhibition was observed with serum from one patient with allergy to latex. CONCLUSIONS: Patients with allergy to plant-derived food and associated pollinosis showed a high frequency of IgE reactivity to Bet v 2, which may cause positive serum IgE determinations to latex and birch pollen due to the presence of cross-reactive epitopes. IgE reactivity to Bet v 2 may serve as an indicator of broad sensitization.

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[70] - Hemmens VJ, Baldo BA, Elsayed S, Bass D. Allergic response to birch and alder pollen allergens influenced by geographical location of allergic subjects. Int Arch Allergy Appl Immunol 1988;87:321-328

A detailed analysis was made of the reactivity patterns of birch pollen-allergic subjects from Norway and Australia to the various IgE-binding components of pollens from several different birch and alder species. Typically, with each of the pollens examined, the Norwegian subjects exhibited a specific limited response pattern in which only a few of the 18 possible allergenic bands were recognized. A single, major IgE-binding band of approximate molecular weight 16-17 kD was recognized in each pollen by all the Norwegian patients used in the study. In contrast to this situation, there was no 'typical' recognition pattern amongst the Australian patients. Although recognition of the 16- to 17-kD components occurred in some cases, it was not dramatically superior to any other single component. Recognition of higher molecular weight components occurred more frequently than with the Norwegian group. Reasons for the differences in the response patterns of the Australian and Norwegian groups are discussed with reference to the exposure of the populations to different allergenic sources and genetic variation

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[71] - Movérare R, Westritschnig K, Svensson M, Hayek B, Bende M, Pauli G, et al. Different IgE Reactivity Profiles in Birch Pollen-Sensitive Patients from Six European Populations Revealed by Recombinant Allergens: An Imprint of Local Sensitization. Int Arch Allergy Immunol 2002;128:325-335

Background: Sensitivity to birch pollen allergens is a common feature among European patients with seasonal pollen allergy. In this in vitro study, we examined the specific serum IgE binding profiles to individual birch pollen allergens in birch-sensitive patients from six European populations. Methods: The study included 242 patients from Finland, Sweden, Austria, France, Switzerland and Italy. All suffered from seasonal rhinoconjunctivitis and/or asthma. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP System and immunoblotting. Results: Almost all Finnish, Swedish and Austrian sera contained IgE specific for Bet v 1 (98%). Bet v 1-specific IgE antibodies were found in 90% of the French sera, and in 65 and 62% of the sera from Switzerland and Italy, respectively. Few Finnish (2%) and Swedish (12%) patients had IgE to Bet v 2, while Bet v 2 reactivity was more common in the other populations (20-43%). Reactivity to Bet v 4 was rare in all populations (5-11%) except for the Italian patients, in whom 3 of 11 sera were positive (27%). The immunoblot results supported the specific IgE profiles obtained with Pharmacia CAP System showing a broader IgE reactivity profile in patients from central and southern Europe as compared to northern Europe. Conclusion: Component-resolved allergy diagnosis with recombinant allergens reveals that the IgE reactivity profiles to individual birch pollen allergens vary between European populations. This observation may be explained by sensitization to different allergen sources and will have an impact on allergen-specific prevention and therapy strategies.

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[72] - Egger C, Focke M, Bircher A, Scherer K, Valenta R. Primary sensitisation to birch pollen in patients allergic to beech and oak pollen. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°277

Background and aims. Allergy to beech and oak pollen is frequently observed in northern and middle Europe and North America. Aim of our study was to investigate the allergen profile of beech and oak. Methods. Sera from tree pollen allergic Swiss patients from an area with low birch pollen exposure (n=23) and Austrian patients with high birch pollen exposure (n=26) were analysed by immunoblot experiments and ELISA for IgE reactivity to birch (Betula pendula), beech (Fagus sylvatica) and oak (Quercus alba) pollen allergens. In addition, IgE inhibition experiments were performed with recombinant and purified natural allergens and allergen specific antibodies were used to identify beech and oak pollen allergens. Results. Preincubation of sera with purified birch and grass pollen allergens (rBet v 1, rBet v 2, rPhl p 7 and nPhl p 4) identified allergens in beech and oak pollen which cross-react with Bet v 1 (17 kDa), profilin (14 kDa), a 2 EF-hand calcium-binding allergen (8 kDa) and Phl p 4 (> 35 kDa). These results were confirmed with allergen-specific antibodies. The majority (20/23) of the Swiss patients and all Austrian patients reacted with Bet v 1. Profilin was recognised by 6 Swiss and 6 Austrian patients, one Austrian patient showed reactivity to the 2 EF-hand allergen. Phl p 4-related immunoreactivity in beech and oak pollen was common in Swiss and Austrian patients. Quantitative assessment of IgE levels by ELISA showed highest IgE values for birch followed by beech and oak pollen in Austrian and Swiss patients. Conclusions. Birch, beech and oak pollen contain cross-reactive allergens. Allergy to beech and oak pollen is mainly due to sensitisation to crossreactive birch pollen allergens. This should be considered when choosing immunotherapy for the treatment of tree pollen allergy.

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[73] - 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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[74] - Karamloo F, Schmitz N, Scheurer S, Foetisch K, Hoffmann A, Haustein D, et al. Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins. J Allergy Clin Immunol 1999;104:991-999

Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.

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[75] - de Amici M, Mosca M, Vignini M, Quaglini S, Moratti R. Recombinant birch allergens (Bet v 1 and Bet v 2) and the oral allergy syndrome in patients allergic to birch pollen. Ann Allergy Asthma Immunol 2003;91:490-492

BACKGROUND: IgE cross-reactivity between pollen and food allergens represents the molecular basis for oral allergy syndrome (OAS). OBJECTIVE: To evaluate specific IgE for Bet v 1 and Bet v 2 in the serum of patients sensitized to birch pollen and to identify whether IgE antibodies to Bet v 1 and Bet v 2 were predictors of OAS. METHODS: Thirty-three patients with skin prick test results and radioallergosorbent assay test results positive to birch pollen, 12 (36%) of whom had OAS symptoms, were enrolled in the study. Serum levels of specific IgE were determined by the fluoroenzyme immunoassay technique. RESULTS: The t test revealed significantly higher serum IgE levels against Bet v 1, Bet v 2, and birch pollen in the 12 symptomatic patients with respect to those without OAS (32.4 vs 12.4 kU/L, 7.6 vs 1.3 kU/L, and 42.3 vs 17.3 kU/L, respectively). Attempts to establish a threshold value of serum IgE antibirch pollen and the appearance of OAS revealed that a level of 20 kU/L or more yields an efficiency of the test equal to 70%. CONCLUSIONS: In our study, quantitative birch specific IgE level proved useful in predicting clinical allergy symptoms with birch exposure.

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[76] - Schierz J, Burow G. Determination of antibody pattern with recombinant allergen components in pollen allergic patients. Allergy Clin Immunol Int 2005;17(Suppl. 1):205

Quantitative determination of specific IgE antibodies to recombinant allergen components allows to establish the individual sensitization profiles of allergic patients. The presence of IgE antibodies to crossreactive components can be used to predict sensitization to related allergen sources. The aim of the study is to determine the IgE antibody pattern in sera of grass and birch pollen allergic patients. Methods: Serum samples from 151 patients were analyzed for IgE antibodies to recombinant major allergens of timothy and birch, Phl p1, Phl p5, Bet v1, the profilins Phl p12, Bet v2, and the calcium-binding proteins Phl p7 and Bet v4 with UniCAP 100 System and the recombinant ImmunoCAP allergens (Pharmacia Diagnostics, Freiburg, Germany). Serum samples with levels >5kU/l to timothy and/or birch pollen were selected. Results: Serum samples of 44 patients were positive for the major allergens of both timothy and birch pollen, Phl p1, Phl p5, Bet v1, and negative for Phl p7, Phl p12, Bet v2 and Bet v4. 51 samples were only positive for Phl p1 and Phl p5, negative for birch, 30 samples were only positive for Bet v1, negative for timothy. 6 samples positive for timothy, levels between 5.7-9.4 kU/l were only positive for the additionally tested component Phl p4. One sample positive for timothy and rye pollen was only positive for Phl p7 and Phl p12, but negative for the major components. In 19 samples IgE antibodies to both timothy and birch pollen major allergens but also to profilins and calcium-binding proteins could be detected, partly with lower levels for the major allergen components. Conclusion: In 125 of 151 samples IgE antibodies were found to only the major allergen components of timothy and/or birch pollen. In 26 samples antibodies to profilins and/or calcium-binding proteins were detected, partly with negative or lower levels for the major allergens. The measurement of IgE antibodies with recombinant allergen components is useful for the detection of individual sensitization pattern to predict crossreactivity and might be of importance for the selection of the patients for specific immunotherapy.

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[77] - Ebo DG, Hagendorens MM, Bridts CH, de Clerck LS, Stevens WJ. Sensitization to cross-reactive carbohydrate determinants and the ubiquitous protein profilin: mimickers of allergy. Clin Exp Allergy 2004;34:137-144

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[78] - Wensing M, Akkerdaas JH, van Leeuwen A, Stapel SO, Bruijnzeel-Koomen CAFM, Aalberse RC, et al. IgE to Bet v 1 and profilin: Crossreactivity patterns and clinical relevance. J Allergy Clin Immunol 2002;110:435-442

BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants . OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance . METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition . RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon . CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.

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[79] - 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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[80] - Scheiner O, Aberer W, Ebner C, Ferreira F, Hoffmann-Sommergruber K, Hsieh LS, et al. Cross-reacting allergens in tree pollen and pollen-related food allergy: implications for diagnosis of specific IgE. Int Arch Allergy Immunol 1997;113:105-108

A number of recombinant allergens are by now constituents of devices that can be routinely used for the detection of specific IgE. Therefore, the results of diagnostic procedures using conventional allergen extracts can be compared with those employing selected recombinant allergens. METHODS: Thirty-four sera from patients allergic to birch pollen were tested with the standard t3-CAP and rBet v 1a- and rBet v 2-CAP. cDNA was prepared by RT-PCR using primers according to the N terminus of purified allergens. Expression cDNA libraries were screened with IgE from selected patients. RESULTS: Twenty-four patients allergic to birch pollen showed the same RAST class with t3 as with rBet v 1a; 8 patients differed within 1 RAST class. In addition, 3 patients showed RAST class 3 with rBet v 2. Besides Bet v 1 and Bet v 2, 3 allergens from celery and avocado belonging to highly conserved protein families were cloned and sequenced. CONCLUSIONS: rBet v 1a can be expected to represent an excellent tool for the diagnosis of patients allergic to birch pollen in Central, Northern, and Eastern Europe. Still, a much higher number of patients has to be tested. For their high degree of conservation, further protein families have to be identified to explain cross-reactivities of birch pollen allergens other than Bet v 1 and Bet v 2 with, e.g., allergens from vegetable food.

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[81] - Movérare R, Westritschnig K, Svensson M, Hayek B, Bende M, Pauli G, et al. Different IgE Reactivity Profiles in Birch Pollen-Sensitive Patients from Six European Populations Revealed by Recombinant Allergens: An Imprint of Local Sensitization. Int Arch Allergy Immunol 2002;128:325-335

Background: Sensitivity to birch pollen allergens is a common feature among European patients with seasonal pollen allergy. In this in vitro study, we examined the specific serum IgE binding profiles to individual birch pollen allergens in birch-sensitive patients from six European populations. Methods: The study included 242 patients from Finland, Sweden, Austria, France, Switzerland and Italy. All suffered from seasonal rhinoconjunctivitis and/or asthma. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP System and immunoblotting. Results: Almost all Finnish, Swedish and Austrian sera contained IgE specific for Bet v 1 (98%). Bet v 1-specific IgE antibodies were found in 90% of the French sera, and in 65 and 62% of the sera from Switzerland and Italy, respectively. Few Finnish (2%) and Swedish (12%) patients had IgE to Bet v 2, while Bet v 2 reactivity was more common in the other populations (20-43%). Reactivity to Bet v 4 was rare in all populations (5-11%) except for the Italian patients, in whom 3 of 11 sera were positive (27%). The immunoblot results supported the specific IgE profiles obtained with Pharmacia CAP System showing a broader IgE reactivity profile in patients from central and southern Europe as compared to northern Europe. Conclusion: Component-resolved allergy diagnosis with recombinant allergens reveals that the IgE reactivity profiles to individual birch pollen allergens vary between European populations. This observation may be explained by sensitization to different allergen sources and will have an impact on allergen-specific prevention and therapy strategies.

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[82] - Shirasaki H, Yamamoto T, Koyanagi Y, Watanabe N, Himi T. Detection of Specific IgE Antibodies in Sera of Japanese Birch-Allergic Patients Using Recombinant Allergens Bet v 1, Bet v 2 and Bet v 4. Allergol Int 2008;57:93-96

.Background: Birch pollen is the major allergen in pollinosis in northern Japan. IgE reactivity to individual birch pollen allergens has been shown to differ between populations of birch pollen-allergic patients living in different countries. In this study, we examined the IgE profiles to recombinant birch pollen allergens in birch-sensitive patients living in Sapporo. Methods: This study used the sera of 40 patients with specific IgE toward birch pollen extract. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP SystemTM. Results: Of 40 sera with positive CAP results for natural birch pollen extract, 39 (97.5%) had specific IgE towards Bet v 1; 6 (15%) contained specific IgE against Bet v 2. Bet v 4 reactivity was documented in only one subject (2.5%). Conclusions: The present data suggest that the specific IgE reactivity profiles to birch pollen allergen in birch-sensitive patients in Sapporo correspond to those in Scandinavia, possibly due to the heavy birch pollen exposure in this area. This observation provides useful information for future birch allergen-specific immunotherapy in Japan.

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[83] - Movérare R, Westritschnig K, Svensson M, Hayek B, Bende M, Pauli G, et al. Different IgE Reactivity Profiles in Birch Pollen-Sensitive Patients from Six European Populations Revealed by Recombinant Allergens: An Imprint of Local Sensitization. Int Arch Allergy Immunol 2002;128:325-335

Background: Sensitivity to birch pollen allergens is a common feature among European patients with seasonal pollen allergy. In this in vitro study, we examined the specific serum IgE binding profiles to individual birch pollen allergens in birch-sensitive patients from six European populations. Methods: The study included 242 patients from Finland, Sweden, Austria, France, Switzerland and Italy. All suffered from seasonal rhinoconjunctivitis and/or asthma. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP System and immunoblotting. Results: Almost all Finnish, Swedish and Austrian sera contained IgE specific for Bet v 1 (98%). Bet v 1-specific IgE antibodies were found in 90% of the French sera, and in 65 and 62% of the sera from Switzerland and Italy, respectively. Few Finnish (2%) and Swedish (12%) patients had IgE to Bet v 2, while Bet v 2 reactivity was more common in the other populations (20-43%). Reactivity to Bet v 4 was rare in all populations (5-11%) except for the Italian patients, in whom 3 of 11 sera were positive (27%). The immunoblot results supported the specific IgE profiles obtained with Pharmacia CAP System showing a broader IgE reactivity profile in patients from central and southern Europe as compared to northern Europe. Conclusion: Component-resolved allergy diagnosis with recombinant allergens reveals that the IgE reactivity profiles to individual birch pollen allergens vary between European populations. This observation may be explained by sensitization to different allergen sources and will have an impact on allergen-specific prevention and therapy strategies.

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[84] - 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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[85] - de Amici M, Mosca M, Vignini M, Quaglini S, Moratti R. Recombinant birch allergens (Bet v 1 and Bet v 2) and the oral allergy syndrome in patients allergic to birch pollen. Ann Allergy Asthma Immunol 2003;91:490-492

BACKGROUND: IgE cross-reactivity between pollen and food allergens represents the molecular basis for oral allergy syndrome (OAS). OBJECTIVE: To evaluate specific IgE for Bet v 1 and Bet v 2 in the serum of patients sensitized to birch pollen and to identify whether IgE antibodies to Bet v 1 and Bet v 2 were predictors of OAS. METHODS: Thirty-three patients with skin prick test results and radioallergosorbent assay test results positive to birch pollen, 12 (36%) of whom had OAS symptoms, were enrolled in the study. Serum levels of specific IgE were determined by the fluoroenzyme immunoassay technique. RESULTS: The t test revealed significantly higher serum IgE levels against Bet v 1, Bet v 2, and birch pollen in the 12 symptomatic patients with respect to those without OAS (32.4 vs 12.4 kU/L, 7.6 vs 1.3 kU/L, and 42.3 vs 17.3 kU/L, respectively). Attempts to establish a threshold value of serum IgE antibirch pollen and the appearance of OAS revealed that a level of 20 kU/L or more yields an efficiency of the test equal to 70%. CONCLUSIONS: In our study, quantitative birch specific IgE level proved useful in predicting clinical allergy symptoms with birch exposure.

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[86] - Rossi RE, Monasterolo G, Monasterolo S. Detection of specific IgE antibodies in the sera of patients allergic to birch pollen using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles. Allergy 2003;58:929-932

BACKGROUND: Birch pollen is a significant cause of immediate hypersensitivity among susceptible subjects in temperate climates, affecting 5-54% of the population in western Europe. We examined the specific serum IgE antibodies towards recombinant allergens Bet v 1, Bet v 2 and Bet v 4 in birch-sensitive patients from the province of Cuneo, north-west Italy . METHODS: Sera were obtained from 372 patients with symptomatic birch pollen-induced allergic rhinitis and/or asthma. A subgroup of these patients suffered from oral allergy syndrome after eating apple. Their sera were evaluated for specific IgE against natural birch pollen and apple extract, as well as Bet v 1, Bet v 2 and Bet v 4 using Pharmacia CAP system (Pharmacia, Uppsala, Sweden) . RESULTS: Of 372 patients 215 (57.80%) had serum-specific IgE towards Bet v 1. A total of 166 sera (44.62%) contained serum-specific IgE to Bet v 2, while Bet v 4 IgE reactivity was documented in 35 subjects (9.41%). Moreover, 146 (39.25%) patients were monosensitized to Bet v 1; 96 (25.81%) patients were monosensitized to Bet v 2; only four sera (1.08%) contained specific IgE towards Bet v 4. Thirty-nine sera (11.02%) did not contain specific IgE to these individual birch pollen allergens. Of course, all 372 sera (100%) had specific IgE against natural birch pollen extract, of which 162 (43.55%) contained specific IgE to apple extract (75.35% of Bet v 1 positive sera) . CONCLUSION: In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen-sensitive patients. Therefore, for a more precise IgE profile of patients allergic to birch, further purified birch pollen allergens (i.e. Bet v 6, Bet v 7, Bet v 8) will be required.

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[87] - Metz-Favre C, Birba E, de Blay F, Pauli G. Que nous apporte le dosage de l'allergène recombinant rBet v 1 dans la prise en charge de la pollinose printannière ? Rev Fr Allergol Immunol Clin 2004;44:357

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[88] - Mari A, Wallner M, Ferreira F. Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4). Clin Exp Allergy 2003;33:1419-1428

BACKGROUND: Birch allergy is one of the most common pollinosis in areas where exposure to high levels of birch pollen is common. Little is known about birch sensitivity in areas without birch pollen exposure and reactivity to birch-related species within the Fagales order . OBJECTIVE: the aim was to evaluate Fagales reactivity within a population not exposed to birch pollen using epidemiological, diagnostic, and laboratory approaches by means of extracts and allergenic molecules . METHODS: A cohort of 5335 respiratory allergic patients was screened by means of skin testing birch, hazel, and oak pollen extracts. Patients were from a birch-free area, but exposed to other Fagales pollen species. A subset of patients was from an intensively cultivated hazel area. A sample of the Fagales allergic population was tested with other Fagales pollen extract (alder, hornbeam, beech, chestnut) and with apple and hazelnut. IgE detection was performed with birch, hazel, oak, apple, and hazelnut extracts, and with Bet v 1, Bet v 2, Bet v 4, and bromelain. IgE immunoblots were performed using birch and hazel extracts. Epidemiological, clinical, and laboratory data were analysed by stratifying the allergic population . RESULTS: Twenty-five percent of the pollen allergic cohort was skin test positive to at least one of the three Fagales species. Combined reactivity to the three species was recorded in 80% of this cohort. Isolated hazel pollen reactivity was recorded in 13.5% of the Fagales allergic patients. Sixty-six percent of these subjects were from the intensively cultivated hazel area. Reactivity to apple and hazelnut was detected by skin test (40%) and IgE reactivity (60%), but only 19% of the positive patients reported symptoms related to at least one of the two foods. Reactivity to Bet v 1 was recorded in 84% of the birch/hazel/oak co-reactivity group, and in 28% of the subjects with the same co-reactivity, but associating a multiple pollen sensitization. IgE to Bet v 2 (50%) and Bet v 4 (23%) panallergens were recorded positive in the latter subset. Bet v 1 prevalence ranged between 48% and 21% among subgroups of patients coming from different areas. Furthermore, an IgE reactivity to hazel-restricted allergenic components was detected among subjects coming from the same area and having a hazel isolated reactivity . CONCLUSION: Fagales allergy can be found in birch-free areas caused by the exposure to other Fagales species. Birch allergens can be useful for mimicking the allergenic extract, but are also the exclusive tools for a fine diagnostic and epidemiological approach to Fagales pollen allergy. Allergenic molecules from the hazel family will increase the panel of available reagents for the molecule-based approach to allergy diagnosis and therapy.

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[89] - Rossi RE, Monasterolo G, Monasterolo S. Detection of specific IgE antibodies in the sera of patients allergic to birch pollen using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles. Allergy 2003;58:929-932

BACKGROUND: Birch pollen is a significant cause of immediate hypersensitivity among susceptible subjects in temperate climates, affecting 5-54% of the population in western Europe. We examined the specific serum IgE antibodies towards recombinant allergens Bet v 1, Bet v 2 and Bet v 4 in birch-sensitive patients from the province of Cuneo, north-west Italy . METHODS: Sera were obtained from 372 patients with symptomatic birch pollen-induced allergic rhinitis and/or asthma. A subgroup of these patients suffered from oral allergy syndrome after eating apple. Their sera were evaluated for specific IgE against natural birch pollen and apple extract, as well as Bet v 1, Bet v 2 and Bet v 4 using Pharmacia CAP system (Pharmacia, Uppsala, Sweden) . RESULTS: Of 372 patients 215 (57.80%) had serum-specific IgE towards Bet v 1. A total of 166 sera (44.62%) contained serum-specific IgE to Bet v 2, while Bet v 4 IgE reactivity was documented in 35 subjects (9.41%). Moreover, 146 (39.25%) patients were monosensitized to Bet v 1; 96 (25.81%) patients were monosensitized to Bet v 2; only four sera (1.08%) contained specific IgE towards Bet v 4. Thirty-nine sera (11.02%) did not contain specific IgE to these individual birch pollen allergens. Of course, all 372 sera (100%) had specific IgE against natural birch pollen extract, of which 162 (43.55%) contained specific IgE to apple extract (75.35% of Bet v 1 positive sera) . CONCLUSION: In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen-sensitive patients. Therefore, for a more precise IgE profile of patients allergic to birch, further purified birch pollen allergens (i.e. Bet v 6, Bet v 7, Bet v 8) will be required.

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[90] - Wensing M, Akkerdaas JH, van Leeuwen A, Stapel SO, Bruijnzeel-Koomen CAFM, Aalberse RC, et al. IgE to Bet v 1 and profilin: Crossreactivity patterns and clinical relevance. J Allergy Clin Immunol 2002;110:435-442

BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants . OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance . METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition . RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon . CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.

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[91] - Schierz J, Burow G. Determination of antibody pattern with recombinant allergen components in pollen allergic patients. Allergy Clin Immunol Int 2005;17(Suppl. 1):205

Quantitative determination of specific IgE antibodies to recombinant allergen components allows to establish the individual sensitization profiles of allergic patients. The presence of IgE antibodies to crossreactive components can be used to predict sensitization to related allergen sources. The aim of the study is to determine the IgE antibody pattern in sera of grass and birch pollen allergic patients. Methods: Serum samples from 151 patients were analyzed for IgE antibodies to recombinant major allergens of timothy and birch, Phl p1, Phl p5, Bet v1, the profilins Phl p12, Bet v2, and the calcium-binding proteins Phl p7 and Bet v4 with UniCAP 100 System and the recombinant ImmunoCAP allergens (Pharmacia Diagnostics, Freiburg, Germany). Serum samples with levels >5kU/l to timothy and/or birch pollen were selected. Results: Serum samples of 44 patients were positive for the major allergens of both timothy and birch pollen, Phl p1, Phl p5, Bet v1, and negative for Phl p7, Phl p12, Bet v2 and Bet v4. 51 samples were only positive for Phl p1 and Phl p5, negative for birch, 30 samples were only positive for Bet v1, negative for timothy. 6 samples positive for timothy, levels between 5.7-9.4 kU/l were only positive for the additionally tested component Phl p4. One sample positive for timothy and rye pollen was only positive for Phl p7 and Phl p12, but negative for the major components. In 19 samples IgE antibodies to both timothy and birch pollen major allergens but also to profilins and calcium-binding proteins could be detected, partly with lower levels for the major allergen components. Conclusion: In 125 of 151 samples IgE antibodies were found to only the major allergen components of timothy and/or birch pollen. In 26 samples antibodies to profilins and/or calcium-binding proteins were detected, partly with negative or lower levels for the major allergens. The measurement of IgE antibodies with recombinant allergen components is useful for the detection of individual sensitization pattern to predict crossreactivity and might be of importance for the selection of the patients for specific immunotherapy.

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[92] - Focke M, Marth K, Valenta R. Molecular composition and biological activity of commercial birch pollen allergen extracts. Eur J Clin Invest 2009;39:429-436

Background Commercial extracts used for diagnosis and treatment of allergy are currently prepared from natural allergen sources. The aim of this study was to analyse birch pollen allergen extracts produced for in vivo diagnosis of birch pollen allergy regarding their contents of individual birch pollen allergens (Bet v 1, Bet v 2 and Bet v 4). Methods Protein contents were measured and the allergen composition was analysed by immunoblotting using antibody probes specific for Bet v 1, Bet v 2 and Bet v 4 in birch pollen extracts from five manufacturers of allergen extracts. The contents of the major birch pollen allergen, Bet v 1, were quantified with a specific two-site binding enzyme-linked immunosorbent assay with nanogram sensitivity for Bet v 1. The biological activities of the allergen extracts were evaluated by skin prick testing in birch pollen allergic patients and compared with their sensitization profiles. Results A more than 10-fold variation regarding total protein contents (23.1-314 mug mL(-1)) and also regarding the amounts of the major birch pollen allergen, Bet v 1 (1.62-19.6 mug mL(-1)) was found. The highly cross-reactive Bet v 4 allergen was absent in three of the five tested extracts. Furthermore, varying skin test results were obtained in birch pollen allergic patients with the allergen extracts. Conclusions Commercial birch pollen extracts exhibit a considerable variability regarding allergen contents and hence deliver varying in vivo test results. These problems might be overcome with recombinant allergen-based preparations.

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[93] - Ebner C, Ferreira F, Hoffmann K, Hirschwehr R, Schenk S, Szepfalusi Z, et al. T cell clones specific for Bet v I, the major birch pollen allergen, crossreact with the major allergens of hazel, Cor a I, and alder, Aln g I. Mol Immunol 1993;30:1323-1329

Tree pollens are responsible for type I allergies during the flowering season in spring. Pollens from birch, hazel and alder constitute the most important allergen sources in this respect in the northern hemisphere Human IgE antibodies, specific for the major allergens of these pollens, are known to crossreact, and in general every tree pollen allergic patient is sensitized to these three pollen allergens. In this study we investigated eight T-helper cell clones (CD3+, CD4+, TCR alpha/beta) with specificity for Bet v I, the major birch pollen allergen, as proved by reactivity with purified natural as well as with recombinant allergen. The T cell clones were used to investigate common T cell epitopes of the Bet v I molecule with Cor a I, the major allergen of hazel pollen and Aln g I, the major allergen of alder pollen. All eight T cell clones reacted with all three proteins with different intensity. Moreover, three T cell clones, which were known to react with immunodominant T cell epitopes on the Bet v I molecule, were tested for reactivity with dodecapeptides synthesized according to the corresponding homologous regions of the Cor a I and Aln g I sequence. All the peptides induced strong T cell proliferation, indicating the existence of multiple cross-reacting epitopes. These findings will have an impact on the production of vaccines for immunotherapy of tree pollen allergies.

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[94] - Bohle B, Radakovics A, Jahn-Schmid B, Hoffmann-Sommergruber K, Fischer GF, Ebner C. Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level. Eur J Immunol 2003;33:3303-3310

Due to IgE cross-reactivity, birch pollen-allergic individuals frequently develop type I hypersensitivity reactions to celery tuber. We evaluated the T cell response to the major allergen in celeriac, Api g 1, and the cellular cross-reactivity with its homologous major allergen in birch pollen, Bet v 1. Api g 1-specific T cell lines (TCL) and clones (TCC) were established from peripheralblood mononuclear cells of allergic patients. Epitope mapping of Api g 1 with overlapping Api g 1-derived peptides revealed one dominant T cell-activating region, Api g 1(109-126). TCL and TCC generated with Api g 1 cross-reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet v 1 were stronger than to Api g 1. Epitopemapping with Bet v 1-derived peptides revealed that T cells specific for several distinct epitopes distributed over the complete Bet v 1 molecule could be activated by Api g 1. Bet v 1(109-126) was identified as the most important T cell epitope for cross-reactivity with Api g 1. This epitope shares 72% amino acid sequence similarity with the major T cell-activating region of the food allergen, Api g 1(109-126). Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross-reactivity with the major birch pollen allergen. The activation of Bet v 1-specific Th2 cells by Api g 1, in particular outside the pollen season, may have consequences for birch pollen-allergic individuals.

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[95] - Bollen M, Wichers H, Helsper J, Savelkoul H, van Boekel M. Heat stability of PR-10 isoform mixtures, purified from birch, celery and carrot. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°32

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[96] - Bohle B, Zwölfer B, Heratizadeh A, Jahn-Schmid B, Dall Antonia Y, Alter M, et al. Cooking birch pollen–related food: Divergent consequences for IgE- and T cell–mediated reactivity in vitro and in vivo. J Allergy Clin Immunol 2006;118:242-249

BACKGROUND: The major birch pollen allergen Bet v 1 cross-reacts with homologous food allergens, resulting in IgE-mediated oral allergy syndromes (OASs). To avoid this food, allergy allergologists and guidebooks advise patients to consume birch pollen-related foods after heating . OBJECTIVE: We sought to evaluate whether cooked Bet v 1-related food allergens induce IgE- and T cell-mediated reactions in vitro and in vivo . METHODS: Recombinant Bet v 1, Mal d 1 (apple), Api g 1 (celery), and Dau c 1 (carrot) were incubated at increasing temperatures. Protein structures were determined by means of circular dichroism. Mediator release was tested in basophil activation assays. PBMCs and Bet v 1-specific T-cell lines with known epitope specificity were stimulated with native and cooked food allergens. Patients with birch pollen allergy who experienced OAS and the exacerbation of atopic dermatitis (AD) on ingestion of fresh apple, celery, or carrot were retested in double-blind, placebo-controlled food challenges with the respective foods in cooked form . RESULTS: In vitro, cooked food allergens lost the capacity to bind IgE and to induce mediator release but had the same potency to activate Bet v 1-specific T cells as native proteins. In vivo, ingestion of cooked birch pollen-related foods did not induce OAS but caused atopic eczema to worsen . CONCLUSION: T-cell cross-reactivity between Bet v 1 and related food allergens occurs independently of IgE cross-reactivity in vitro and in vivo. In patients with AD, the resulting immune reaction can even manifest as late eczematous skin reactions. Therefore the view that cooked pollen-related foods can be consumed without allergologic consequences should be reconsidered. CLINICAL IMPLICATIONS: Symptom-free consumed pollen-related food allergens might cause T cell-mediated late-phase skin reactions in patients with pollen allergy and AD.

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[97] - 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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[98] - Valenta R, Kraft D. Type I allergic reactions to plant-derived food: a consequence of primary sensitization to pollen allergens. J Allergy Clin Immunol 1996;97:893-895

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[99] - Kazemi-Shirazi L, Pauli G, Purohit A, Spitzauer S, Fröschl R, Hoffmann-Sommergruber K, et al. Quantitative IgE inhibition with purified recombinant allergens indicate pollen-derived allergens as the sensitizing agents responsible for many forms of plant food allergy. J Allergy Clin Immunol 2000;105:116-125

Type I allergic symptoms in the oropharyngeal mucosa upon contact with plant-derived food in patients with pollen allergies have been termed oral allergy syndrome (OAS). IgE cross-reactivity between pollen and food allergens represents the molecular basis for this phenomenon. The sensitizing allergen source (pollen or plant food) in OAS is a controversial issue. OBJECTIVE: We sought to determine the primary sensitizing molecules in patients with OAS. METHODS: We used recombinant birch pollen (rBet v 1 and rBet v 2) and plant food allergens (apple, rMal d 1; celery, rApi g 1; and carrot, rDau c 1), as well as natural pollen (birch and timothy grass) and plant food (apple, peach, kiwi, hazelnut, celery, and carrot) allergens, to identify cross-reactive allergens by using qualitative immunoblot inhibitions. In addition, we determined the percentage of plant food-specific IgE that can be preadsorbed with recombinant and natural pollen allergens by quantitative RAST inhibitions by using sera from 71 patients with OAS. RESULTS: Preincubation of sera with recombinant and natural pollen allergens led to an almost complete inhibition of IgE binding to plant food allergens in Western blots, as well as in RAST inhibition experiments. In contrast, recombinant plant food allergens poorly inhibited IgE binding to Bet v 1. CONCLUSION: Most IgE epitopes in plant food recognized by patients with OAS are resembled by pollen allergens. Thus pollen allergens may be responsible for the elicitation and maintenance of OAS.

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[100] - 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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[101] - Bolhaar STHP, van Ree R, Bruijnzeel-Koomen CAFM, Vieths S, Hoffmann-Sommergruber K, Knulst AC, et al. Severe Allergy to Sharon Fruit Caused by Birch Pollen. Int Arch Allergy Immunol 2005;136:45-52

BACKGROUND: Allergy to sharon fruit (persimmon) has been only rarely reported. Cross-reactivity with pollen (profilin and Bet v 6) appeared to be involved, but Bet v 1 has not been implicated previously . OBJECTIVE: It is our aim to identify whether Bet v 1 sensitization is linked to sharon fruit allergy . METHODS: Two patients with a reaction upon first exposure to sharon fruit were included in the study, as well as 7 patients with birch-pollen-related apple allergy. Sensitivity was assessed by skin prick testing (SPT), a radio-allergosorbent test (RAST) and immunoblotting. RAST analysis was performed for Bet v 1, Bet v 2 and Bet v 6. Cross-reactivity was evaluated by RAST and immunoblot inhibitions. Biological activity of IgE was measured by basophil histamine release. Sharon fruit allergy was evaluated by double-blind placebo-controlled food challenge (DBPCFC) or open challenge (OC) . RESULTS: Both sharon-fruit-allergic patients demonstrated positive reactions in the RAST (8.6 and 6.2 IU/ml, respectively) and SPT (wheal area 37 and 36 mm2). Sharon fruit allergy was confirmed by DBPCFC in 1 patient. The second patient refused a challenge because of the severe initial reaction. Sera from both patients were reactive to Bet v 1 and Bet v 6, which was cross-reactive with sharon fruit by inhibition assays. The patient with the severest reactions was reactive to profilin on immunoblotting. However, profilin did not induce significant histamine release, nor did Bet v 6. Bet v 1 induce approximately 60% histamine release. An OC with sharon fruit in 7 patients allergic to birch pollen and apple, who had not eaten sharon fruit previously, was positive in 6/7 cases . CONCLUSIONS: Birch-pollen-related allergy to sharon fruit is mediated by the known cross-reactive pollen allergens including Bet v 1 and may become more of a problem should sharon fruit consumption increase.

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[102] - Jensen-Jarolim E, Gerstmayer G, Kraft D, Scheiner O, Ebner H, Ebner C. Serological characterization of allergens in poppy seeds. Clin Exp Allergy 1999;29:1075-1079

BACKGROUND AND OBJECTIVE: Poppy seeds in food can induce immediate-type allergic reactions ranging from mild local symptoms to severe anaphylactic reactions. Previous publications showed that poppy seeds cross-react with other plant-derived allergens. The IgE-binding components have not been defined so far. METHODS: We analysed sera from 11 patients with adverse reactions after ingestion of poppy seed-containing food by IgE-immunoblotting. Nine of 11 patients showed concomitant IgE binding to allergens of birch, mugwort or grass pollen in RAST-CAP, and suffered from characteristic seasonal symptoms. RESULTS: Ten of 11 patients showed IgE binding to a 45-kDa protein, 4/11 to a 34-kDa, 5/11 to a 17-kDa, 5/11 to a 14-kDa, and 3/11 to a 5-kDa component. Furthermore, individual IgE binding to proteins of 20, 25, 30 and 40 kDa proteins could be observed. Periodate treatment of blots markedly reduced the IgE binding capacity of the 40- and 45-kDa compounds, indicating the existence of IgE epitopes of the carbohydrate type. Inhibition studies indicated the presence of homologues of pollen allergens in extracts from poppy seeds, i.e. Bet v 1 and Bet v 2. CONCLUSION: The serological analysis showed IgE binding to protein and sugar components of poppy seeds. The 40- and 45-kDa allergens are glycoproteins and contain IgE binding carbohydrate moieties. Moreover, cross-reacting homologues of pollen allergens including Bet v 1 and profilin were detected in poppy seed extract.

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[103] - Bolhaar STHP, van Ree R, Bruijnzeel-Koomen CAFM, Knulst AC, Zuidmeer L. Allergy to jackfruit: a novel example of Bet v 1-related food allergy. Allergy 2004;59:1187-1192

Rationale Confirm allergy to jackfruit in 2 patients with an according personal history and characterize allergens and cross-reactivity of specific IgE antibodies to (birch) pollen and food allergens. In addition, determine the prevalence of jackfruit allergy in a group (n=5) of birch pollen (BP) and food allergic patients. Method s : All patients underwent skin prick testing (SPT). Two patients were tested in a double-blind placebo-controlled food challenge (DBPCFC) and the additional 5 underwent an open challenge (OC) with jackfruit. Specific IgE was measured with the radio-allergosorbent test (RAST). RAST and immunoblot (inhibitions) were carried out. In the 2 jackfruit allergic patients, basophil histamine release was measured with several allergens. Result s : Both patients had positive SPT responses to jackfruit and BP and reacted with oral allergy symptoms (OAS) in the DBPCFC. The 5 selected patients reacted with OAS in the OC. Specific IgE of patient 1 to jackfruit was completely inhibited by BP extract and natural (n)Bet v 1 in RAST and immunoblot inhibition. In addition, this patient showed histamine release upon stimulation with jackfruit, apple or BP extracts and nBet v 1. Conclusions : Sensitization to jackfruit allergy is due to the cross-reactive BP allergen Bet v 1. The prevalence of jackfruit allergy in the selected group of 5 patients was 100%, but jackfruit is not regularly consumed in Northern Europe, explaining why jackfruit allergy is not common.

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[104] - Hemmer W, Focke M, Marzban G, Laimer M, Jarisch R. Identification of fig and other moraceae fruits as new birch pollen-associated foods. Allergy 2009;64(Suppl. 90):164

Background: Allergy to fig fruit (Ficus carica) has been described in patients with sensitization to Ficus benjamina and rubber latex. We investigated the cross-reactivity of fig fruit and other fruits from the Moraceae family with birch pollen allergens. Methods: 164 patients with or without birch pollen allergy were tested by prick-to-prick test with fig fruit, mulberry, and jackfruit (family Moraceae) as well as with various other foods known to be associated with birch pollen allergy. Allergenic extracts from fig, mulberry, and jackfruit were separated by SDS-PAGE and tested with patient sera and polyclonal antisera against Bet v 1 and Mal d 1. Western blot inhibition was carried out with Moraceae fruit extracts, birch pollen extracts, and recombinant Bet v 1. Results: Among 87 patients with isolated birch pollen allergy, 73% had a positive skin test to fresh fig, 12% to dried fig, 89% to mulberry, 90% to jackfruit, 80% to Rosaceae fruits, and 80% to hazelnut. In 59 patients with isolated Ficus benjamina sensitization, the reactivity rates to the same foods were 80 - 34 - 5 - 7 - 1 - 0%. None of 18 patients with monovalent mugwort pollen sensitization reacted to the Moraceae fruits. 62% of birch pollen-sensitized patients testing positive for fig fruit reported adverse reactions after consumption of fresh figs (mostly oral allergy syndrome), whereas dried figs were generally well tolerated. Antibody-binding of polyclonal antisera and patient sera to a double band around 17 kDa in all blotted Moraceae fruits was specifically inhibited by birch pollen extracts and rBet v 1. Conclusion: Fresh fig and other Moraceae fruits contain allergens with homology to Bet v 1 and represent clinically relevant birch pollen-associated foods. Allergens cross-reactive with Ficus benjamina (cysteine proteases) occur in fresh and dried fig but not in other Moraceae fruits.

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[105] - Mittag D, Akkerdaas J, Ballmer-Weber BK, Vogel L, Wensing M, Becker WM, et al. Ara h 8, a Bet v 1–homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy. J Allergy Clin Immunol 2004;114:1410-1417

Background We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. Objective : We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1ˆhomologous peanut allergen Ara h 8. Method s : Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. Result s : During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8ˆspecific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. Conclusions : Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.

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[106] - 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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[107] - Ballmer-Weber BK, Hoffmann A, Wüthrich B, Lüttkopf D, Pompei C, Wangorsch A, et al. Influence of food processing on the allergenicity of celery: DBPCFC with celery spice and cooked celery in patients with celery allergy. Allergy 2002;57:228-235

Background:Celery root is often consumed in a processed form as a cooked vegetable or as a spice. So far, however, there has been no information about the allergenicity of processed celery in celery-allergic patients. Methods:In 12 patients with a history of allergic reactions to raw or raw and cooked celery, double-blind placebo-controlled food challenges (DBPCFCs) with raw celery (n=10), cooked celery (110°C/15min; n=11), and celery spice (n=5) were performed. Nine patients underwent an open mucosal challenge with four samples of canned celery retorted at Co-values (cooking effect) of 7.45-76.07 (corresponding to the time periods in minutes at a thermal influence of 100°C). IgE immunoblot analysis of celery extract was performed with sera of all challenged patients. The thermal stability of celery allergen was investigated by enzyme allergosorbent test (EAST) inhibition. Furthermore, intraperitoneal immunization of mice followed by a rat basophil leukemia (RBL) cell mediator release assay was used as a biological in vitro model to assess the allergenicity of processed celery. Results:Six out of 11 patients showed a positive DBPCFC to cooked celery and five out of five patients to celery spice. Allergenicity of celery was preserved in four patients with a positive DBPCFC to cooked celery even if celery was treated at a Co-value of 76.07. Patients with positive DBPCFC to cooked celery reacted to known celery allergens (Apig1, Apig4, cross-reactive carbohydrate determinants CCD). EAST inhibition showed that heat resistance of celery allergens decreases in the following order: CCD>Apig4>Apig1. Accordingly, five of six patients with a positive DBPCFC to cooked celery were sensitized to profilin and/or CCD. The murine model reflected the reactivity of patients sensitized to the major allergen Apig1. Conclusions:1) In a subset of patients with a positive DBPCFC to cooked celery, celery remains allergenic even after extended thermal treatment (76.07 min/100°C). 2) Celery spice is allergenic for patients with an allergy to raw celery. 3) RBL cells sensitized with mouse IgE to raw celery may serve as a useful tool for screening the potential allergenicity of heat-processed products containing celery

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[108] - Vieths S, Scheurer S, Ballmer-Weber B. Current Understanding of Cross-Reactivity of Food Allergens and Pollen. Ann N Y Acad Sci 2002;964:47-68

-Pollen-allergic patients frequently present allergic symptoms after ingestion of several kinds of plant-derived foods. The majority of these reactions is caused by four distinct cross-reactive structures that are present in birch pollen. Proteins that share common epitopes with Bet v 1, the major birch pollen allergen, occur in pollens of several tree species: apples, stone fruits, celery, carrot, nuts, and soybeans. Approximately 70% of our patients who are allergic to birch pollen may experience symptoms after consumption of foods from these groups. In contrast, two minor allergenic structures-profilins and cross-reactive carbohydrate determinants (CCD)-that sensitize approximately 10-20% of all pollen-allergic patients are also present in grass pollen and weed pollen. Moreover, IgE-binding proteins related to the birch pollen minor allergen Bet v 6 have been found in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini, and carrot. Frequently, the occurrence of cross-reactive IgE antibodies is not correlated with the development of clinical food allergy. In particular, the clinical relevance of sensitization to CCD is doubtful. Generally, pollen-related allergens tend to be more labile during heating procedures and in the digestive tract compared to allergens from classical allergenic foods such as peanut. However, recent DBPCFC studies have shown that both cooked celery and roasted hazelnuts still pose an allergenic risk for pollen-sensitized subjects. Since pathogenesis-related proteins share several common features with allergens and both the Bet v 1 and the Bet v 6-related food allergens are defense-related proteins, approaches to introduce such proteins as a measure to protect plants against diseases should be performed with caution as they may increase the allergenicity of these crops

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[109] - Mittag D, Akkerdaas J, Ballmer-Weber BK, Vogel L, Wensing M, Becker WM, et al. Ara h 8, a Bet v 1–homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy. J Allergy Clin Immunol 2004;114:1410-1417

Background We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. Objective : We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1ˆhomologous peanut allergen Ara h 8. Method s : Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. Result s : During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8ˆspecific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. Conclusions : Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.

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[110] - Burnett GR, Wickham M, Fillery-Travis A, Robertson JA, Belton PS, Gilbert SM, et al. Interaction between protein allergens and model gastric emulsions. Biochem Soc Trans 2002;30:916-918

The observed resistance to pepsinolysis of known food allergens has been suggested as a predictor of their allergenic risk. Consequently, resistance to pepsinolysis has become incorporated into decision tree assessment for potential allergenic risk posed by novel foods. However, existing methods take little account of the interaction between food structure and physiological conditions existing during digestion in vivo. Here we show that a range of protein allergens can adsorb to model stomach emulsions, providing a further means of resisting digestion. We also show that raising the pH and the addition of bile salts to a model stomach emulsion, thereby mimicking the duodenal environment, has the effect of desorbing the adsorbed protein.

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[111] - Frémont S, Sanchez C, Errahali Y, Mouécoucou J, Metche M, Méjean L, et al. Allergénicité alimentaire: importance des interactions protéines-lipides et protéines-polysaccharides. Alim'Inter 2003;8:35-41

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[112] - Sancho A, Wangorsch A, Jensen B, Johnson P, Watson A, Alexeev Y et al. The Bet v 1 scaffold is inherently unstable to low pH and proteolysis. Allergy 2009;64(Suppl. 90):369

Background: More than 50% of birch pollen-allergic patients experience adverse reactions after ingestion of certain foods (apple, peach, cherry, carrot, celery, hazelnut, walnut). This is in most cases due to cross-reactivity of the major birch pollen allergen, Bet v 1, to homologous structures identified in these foods that belong to the PR10 protein family. We investigated whether the Bet v 1 scaffold is inherently unstable to low pH and hence susceptible to proteolysis. We also assessed the effect of the surfactant phosphatidyl choline (PC), which is abundant in certain foods and is secreted by the gastric mucosa, on the stability of Bet v 1 homologues. Methods: Bet v 1 homologues from apple, celery, hazelnut and peach were produced as recombinant allergens. Phosphatidyl choline (PC) vesicles were prepared from an egg lecithin solution by sonication in Standard Gastric Fluid (SGF, 0.15M saline solution, pH 2.5). In vitro gastric digestion was performed in SGF with pepsin (60 min, 37°C) in the presence or absence of PC. Interaction of the allergens with PC was assessed by Fluorescence Polarization. Digestion profiles were followed by SDS-PAGE and peptides identified by MS. IgE reactivity of the undigested and digested samples was determined by IgE immunoblotting, ELISA inhibition, and their biological activity by basophil activation tests. Results: All Bet v 1 homologues were susceptible to pH-induced unfolding but showed subtle differences. All were broken down rapidly by pepsin. Addition of PC had a slight protective effect and Mal d 1 was found to be the only allergen to be associated with PC vesicles. IgE binding capacity and basophil activation of all allergens were lost during digestion. Results were consistent with levels of residual intact allergens. Conclusion: These data show that the pH instability and susceptibility to pepsinolysis is determined by the Bet v 1 scaffold and is modulated to only a minor extent by variations in primary sequence. This work is funded by the EC FP6 project EuroPrevall (FOOD-CT-2005-514000).

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[113] - Bilo B, Dyga W, Obtulowicz K. Specific IgE level to Bet v 1 and Bet v 2 and oral allergy syndrome in pollen allergy patients. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1506

Profilin and main birch allergen are among plant allergens associated commonly with pollen allergy and concomitant plant-derived food reactivity (oral allergy syndrome OAS). Aim of the present study was to determine the relationship between OAS and an elevated level of specific immunoglobulin E (slgE) to Bet v 1 and Bet v 2 (profilin) recombinant proteins. Study population included 60 subjects (31 M, 29 F) randomly selected from 123 subjects treated for several years in the Department of Clinical and Environmental Allergology in Cracow who have regularly completed the symptoms` charts in pollen season 2003 or 2004. In all these subjects the following data were obtained: history of symptoms and food reactions; prick skin tests with inhaled and food allergens (Allergopharma - AT) and native tests (NT) with food allergens; levels of total IgE and sIgE to birch (T3), timothy (G6), mugwort (W6), D. pteronissimus (D1) and recombinant Bet v 1 and Bet v 2. Twenty-one subjects reported OAS symptoms (most frequently to celery - 14, hazelnut - 14, and apple - 12 subjects) including throat irritation (13), itching in the mouth (10), oedema of lip (9) or throat (7 subjects). Mean sIgE levels in OAS subjects compared with OAS-free subjects were: Bet v 1 - 33.9 vs. 1.88 kUA/l (p<0.001), T3 - 38.4 vs. 4.3 kUA/l (p<0.001) with no differences in other sIgE levels. OAS subjects had more frequently: allergy to early (mainly hazel tree, p<0.001) and late tree pollen (mainly birch, p<0.001), positive results of skin tests with celery (p<0.001 both AT and NT), hazelnut (AT - p<0.01, NT - p<0.001), peach (p<0.05 both), apple (p<0.001, NT only) and carrots (p<0.005, NT only). Among 26 subjects with elevated sIgE to Bet v 1 as many as 17 developed OAS symptoms (p<0.001), while in profilin sensitive subjects no such association was seen. Polish subjects with tree pollen allergy, with elevated birch sIgE (in particular to main birch allergen) are at a high risk of developing OAS. Elevated sIgE to profilin is not related to an increased rate of plant-derived food intolerance. Fresh celery, hazelnuts and apples intake by subjects allergic to tree pollen seems particularly hazardous. Skin tests with native allergens are necessary for a complete diagnosis of a potential sensitivity to plant-derived foods.

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[114] - de Amici M, Mosca M, Vignini M, Quaglini S, Moratti R. Recombinant birch allergens (Bet v 1 and Bet v 2) and the oral allergy syndrome in patients allergic to birch pollen. Ann Allergy Asthma Immunol 2003;91:490-492

BACKGROUND: IgE cross-reactivity between pollen and food allergens represents the molecular basis for oral allergy syndrome (OAS). OBJECTIVE: To evaluate specific IgE for Bet v 1 and Bet v 2 in the serum of patients sensitized to birch pollen and to identify whether IgE antibodies to Bet v 1 and Bet v 2 were predictors of OAS. METHODS: Thirty-three patients with skin prick test results and radioallergosorbent assay test results positive to birch pollen, 12 (36%) of whom had OAS symptoms, were enrolled in the study. Serum levels of specific IgE were determined by the fluoroenzyme immunoassay technique. RESULTS: The t test revealed significantly higher serum IgE levels against Bet v 1, Bet v 2, and birch pollen in the 12 symptomatic patients with respect to those without OAS (32.4 vs 12.4 kU/L, 7.6 vs 1.3 kU/L, and 42.3 vs 17.3 kU/L, respectively). Attempts to establish a threshold value of serum IgE antibirch pollen and the appearance of OAS revealed that a level of 20 kU/L or more yields an efficiency of the test equal to 70%. CONCLUSIONS: In our study, quantitative birch specific IgE level proved useful in predicting clinical allergy symptoms with birch exposure.

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[115] - Daschner A, Crespo JF, Pascual CY. Specific IgE to recombinant vegetal panallergen (rBet v 2) and fruit allergy in pollinic patients. Allergy 1999;53:614-618

The presence of IgE antibodies to the allergens rBet v 1 and rBet v 2 was investigated in patients with known sensitization to rye grass (Lolium perenne) and/or olive (Olea europaea) pollen, by comparing a group of 10 patients who had allergic symptoms after ingestion of fruits [including bananas, figs, kiwifruit, muskmelons, oranges and peaches] with a group of 17 patients who had only seasonal respiratory symptoms. There was no significant difference between the 2 groups for total IgE. All patients showed specific IgE to both L. perenne and O. europaea. No specific IgE binding to rBet v 1 was detected in any patient. The incidence of the presence of IgE antibodies to rBet v 2 was 90% in the group with fruit allergy and 35% in the group [without fruit allergy]. There was a significant association between presence of IgE antibodies to rBet v 2 and fruit allergy (P = 0.007). Specific IgE values to O. europaea pollen were higher in the fruit-allergy group than the group without fruit allergy (P = 0.032). It is concluded that pollen-allergic patients with specific IgE to birch pollen profilin show a significantly elevated frequency of fruit allergy.

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[116] - 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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[117] - Pastorello EA, Pravettoni V, Farioli L, Ispano M, Fortunato D, Monza M, et al. Clinical role of a lipid transfer protein that acts as a new apple-specific allergen. J Allergy Clin Immunol 1999;104:1099-1106

Allergy to apple is commonly associated with birch pollinosis because the two share homologous allergens. However, some patients have apple allergy but no birch pollinosis, suggesting that there are allergens that do not cross-react with birch. OBJECTIVE: The aim of the study was to evaluate the IgE reactivity pattern to an apple extract in subjects with allergic reactions to apple, with and without birch hay fever. METHODS: Forty-three patients with oral allergy syndrome for apple and positive open food challenge, skin prick test, and serum specific IgE antibodies to apple were admitted to the study. Thirty-two had birch pollinosis (documented by specific IgE for birch) and 11 were not allergic to birch. The IgE reactivity pattern to apple extract was identified by SDS-PAGE and immunoblotting. The consistent allergen, a 9-kd protein, was then purified by HPLC and characterized by periodic acid-Schiff staining, isoelectric point, and N-terminal amino acid sequencing. RESULTS: The sera from 28% of patients allergic to apple with birch pollinosis, but from all patients allergic only to apple, recognized the 9-kd protein. This protein has an isoelectric point of 7.5 and is not glycosylated. Determination of its partial amino acid sequence showed that it belongs to the family of lipid transfer proteins, which act as major allergens in Prunoideae fruits. CONCLUSIONS: These results indicate that a lipid transfer protein is an important allergen in patients allergic to apple but not to birch pollen. The prevalent IgE reactivity to this allergen in subjects with no birch pollinosis and the physicochemical characteristics of this protein suggest that sensitization may occur through the oral route.

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[118] - Stapleton GL, Griffiths H, Karim MY, Deacock SJ. Specific IgE reactivty to birch pollen allergens in a UK population with the oral allergy syndrome: are there any pre-disposing factors for anaphylaxis ? Clin Exp Allergy 2006;36:1210-1211

Introduction Pollen-allergic patients frequently experience symptoms on ingestion of fresh plant-derived foods termed oral allergy syndrome (OAS). IgE cross-reactivity between pollen and food allergens represents the molecular basis of OAS. Symptoms range from commonly immediate oral itching to rarely lifethreatening anaphylaxis. Studies from Europe have described use of recombinant birch pollen allergens to identify birch pollen-sensitive patients and those with OAS; however this has not been evaluated in the UK or for predicting severity of symptoms in OAS. Objectives To determine whether patients with OAS-related anaphylaxis reacted to any particular birch pollen allergen and whether specific IgE (sIgE) level to any particular allergen reflected disease severity. Methods 280 patients with birch pollen sensitivity were analysed retrospectively for associations between clinical food sensitivities (e.g. apple, carrot, hazelnut etc.) and incidence of anaphylaxis. Of 280 patients, 65 serum samples were tested for sIgE to natural birch pollen extract, recombinant (r)Bet v 1, rBet v 2 and rBet v 4 by fluoroenzyme immunoassay using the Pharmacia UniCAPs 100E automated system. Results No correlation was found between specific clinical food sensitivities and incidence of anaphylaxis. No correlation was found between reactivity to particular birch pollen allergens and incidence of anaphylaxis. Likewise, no correlation was found between level of sIgE to birch pollen allergens and incidence of anaphylaxis. Conclusion Measurement of specific IgE to birch pollen allergens could not be used to predict disease severity in OAS. Further work to identify markers for OAS severity is necessary.

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[119] - 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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[120] - 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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[121] - Mothes N, Valenta R. Biology of tree pollen allergens. Curr Allergy Asthma Rep 2004;4:384-390

More than 25% of the population suffer from type I allergy. Pollens from trees of the Fagales, Oleaceae, and Cupressaceae belong to the most potent and frequent allergen sources. During the past 15 years, the nature of the most important allergens has been identified by molecular biological techniques, and recombinant allergens equivalent to the natural allergens have been produced. These advances provide insight into the biological functions of important allergens and allow the development of novel forms of diagnosis and therapy. In this review, we focus on Fagales allergens to illustrate the impact of recombinant allergens on diagnosis and therapy. We discuss structural similarities as a molecular basis for cross-reactivities and develop diagnostic concepts by using species-specific marker allergens as well as highly cross-reactive allergens. The identification of the allergen recognition profiles of patients with recombinant allergens allows a more precise selection of patients for available forms of allergy treatment. Moreover, we describe novel recombinant allergen-based forms of specific immunotherapy.

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[122] - Mothes N, Horak F, Valenta R. Transition from a Botanical to a Molecular Classification in Tree Pollen Allergy: Implications for Diagnosis and Therapy. Int Arch Allergy Immunol 2004;135:357-373

Tree pollens are among the most important allergen sources. Allergic cross-reactivity to pollens of trees from various plant orders has so far been classified according to botanical relationships. In this context, cross-reactivities to pollens of trees of the Fagales order (birch, alder, hazel, hornbeam, oak, chestnut), fruits and vegetables, between pollens of the Scrophulariales (olive, ash, plantain, privet, lilac) and pollens of the Coniferales (cedar, cypress, pine) are well established. The application of molecular biology methods for allergen characterization has revealed the molecular nature of many important tree pollen allergens. We review the spectrum of tree pollen allergens and propose a classification of tree pollen and related allergies based on major allergen molecules instead of botanical relationships among the allergenic sources. This molecular classification suggests the major birch pollen allergen, Bet v 1 as a marker for Fagales pollen and related plant food allergies, the major olive pollen allergen, Ole e 1, as a possible marker for Scrophulariales pollen allergy and the cedar allergens, Cry j 1 and Cry j 2, as potential markers for allergy to Coniferales pollens. We exemplify for Fagales pollen allergy and Bet v 1 that major marker allergens are diagnostic tools to determine the disease-eliciting allergen source. Information obtained by diagnostic testing with marker allergens will be important for the appropriate selection of patients for allergen-specific forms of therapy.

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[123] - Swoboda I, Twaroch T, Valenta R, Grote M. Tree pollen allergens. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 87-105

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[124] - Petersen BN, Janniche H, Munch EP, Wihl JA, Böuwadt H, Ipsen H, et al. Immunotherapy with partially purified and standardized tree pollen extracts. I. Clinical results from a three-year double-blind study of patients treated with pollen extracts either of birch or combinations of alder, birch and hazel. Allergy 1988;43:353-362

Fifty-four adult patients with tree pollen-induced rhinitis (28), asthma (1), or rhinitis and asthma (25) were selected for immunotherapy with standardized and partly purified tree pollen extracts using a double blind protocol. The selection was based on clinical history, results of nasal or bronchial challenge, skin prick tests and RAST. Further, based on crossed radio-immunoelectrophoresis, sex, age and severity of symptoms, the patients were allocated in matched pairs and the treatment alternatives were randomly distributed within the pairs. Twenty-three patients treated with extracts composed of any combination of alder, birch and hazel pollen which matched their IgE response in CRIE (Group 1 (ABC)) and 22 patients receiving birch pollen extracts (Group 2 (B)) completed ail 3 years of treatment. The in vivo results comprising symptom and medicine consumption scores are given here. Changes in specific skin and nasal reactivity as well as in immunological parameters arc presented separately. No significant differences were demonstrated between the treatment groups in the two parameters. Both extracts were effective and reduced in general the symptom scores to one tenth of the starting level. Expressed another way, at the end of the study, the patients tolerated 30 times more pollen until symptoms of the same severity were elicited, compared to before. In the Nordic countries, spring-time asthma and rhino-conjunctivitis caused by pollen from deciduous trees can be effectively treated with an extract of birch pollen alone.

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[125] - Malandain H. IgE-reactive carbohydrate epitopes--classification, cross-reactivity, and clinical impact. Eur Ann Allergy Clin Immunol 2005;37:122-128

A glycan-related IgE-reactivity has been demonstrated in most allergen sources, especially in plant kingdom. Recent progress in glycobiology has allowed a clearer classification of these glyco-epitopes. Unlike classical peptide chain-based epitopes, glyco-epitopes can share significant structural homologies beyond the limits of protein families. These glycoepitopes are thus prone to extensive cross-reactivity. They have been called Cross-reactive Carbohydrate Determinants or CCD. Many of these glyco-epitopes behave as "panepitopes", leading to cross-reactivity between products as distant as pollens and hymenoptera venoms. But CCD are not universally cross-reactive and they rather cluster into subgroups such as plant CCD or fungal CCD. Because a monovalent IgE-binding is sufficient in serum-based assays, glyco-epitopes and CCD are classically considered as a potential source of positive in vitro results without clinical significance. But some authors recently demonstrated that glyco-epitopes could induce a response at the cell level and suggested that they might play a role in vivo. As long as in vitro assays include glycan- and CCD-related IgE responses, laboratory results should be carefully interpreted in the light of existing knowledge about the glycomes of natural products. IgE-reactivity of the patient's serum can also be tested towards a glycoprotein model such as bromelain.

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[126] - Wilson IBH, Harthill JE, Mullin NP, Ashford DA, Altmann F. Core alpha-1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 1998;8:651-661

Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.

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[127] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[128] - Ebo DG, Hagendorens MM, Bridts CH, de Clerck LS, Stevens WJ. Sensitization to cross-reactive carbohydrate determinants and the ubiquitous protein profilin: mimickers of allergy. Clin Exp Allergy 2004;34:137-144

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[129] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[130] - van Ree R, Van Leeuwen WA, Akkerdaas JH, Aalberse RC. How far can we simplify in vitro diagnostics for Fagales tree pollen allergy ? A study with three whole pollen extracts and purified natural and recombinant allergens. Clin Exp Allergy 1999;29:848-855

BACKGROUND: Current diagnostic tests for Fagales tree pollen allergy are often composed of mixtures of pollen of birch, alder and hazel. Their complex composition hampers accurate standardization . OBJECTIVE: The aim of this study was to investigate whether mixtures of tree pollen extracts can be replaced by a single pollen species, and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens . METHODS: Sera (n = 1725) were selected on ground of a general suspicion for inhalant allergy, and tested in a RAST for birch, alder and hazel pollen. Sera with > 0.5 RU/mL for any of the three species were tested in a RAST for natural Bet v 1 and Bet v 2 as well as for recombinant versions of both allergens . RESULTS: Specific IgE antibodies (> 0.3 RU/mL) against birch, alder and hazel were found in 242, 298 and 292 sera, respectively. All sera with a positive RAST for alder and/or hazel and a negative RAST for birch were low-responder sera on alder and hazel, only five sera having a RAST value > 1.0 (all < 2.0). For all sera with a RAST > 0.5 RU/mL (n = 250), the mean of individual ratio's alder/birch and hazel/birch was 1.02 and 0.54, respectively. Of 223 of these sera, 63.2% had specific IgE against natural Bet v 1 and 63.7% against natural Bet v 2. When responses to both allergens were combined 93.7% were positive. The mean ratios Bet v 1 + 2/extract were 1.00, 1.04 and 2. 11 in case of birch, alder and hazel, respectively. For 211 sera the same analysis was performed with recombinant Bet v 1 and Bet v 2. Only six sera with Bet v 1-specific IgE (all < 0.5 RU/mL) were negative (< 0.3 RU/mL) on recombinant Bet v 1. For Bet v 2, 77/132 sera with specific IgE to the natural allergen did not react to the recombinant version. Twelve false-negatives had RAST values > 1.0 RU/mL. The mean of the individual recombinant/natural ratios was 0. 98 for Bet v 1 and 0.38 for Bet v 2 (P < 0.001). The mean ratio rBet v 1 + 2/birch was 0.75 with 17.5% false-negatives on the combination of recombinant allergens . CONCLUSION: Reliable in vitro diagnosis is possible with a single tree pollen extract (birch or alder). The same is true for purified natural Bet v 1 and Bet v 2. A combination of recombinant molecules is slightly less efficient.

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Les allergènes du pollen de bouleau

Les prévalences de réactivité au pollen de bouleau et à ses allergènes

Les réactivités / allergies croisées du pollen de bouleau

Réactivités aux allergènes du bouleau et indication de l’immunothérapie

Pollen de bouleau et CCD

Rubriques

Dans la même rubrique

Les Cupressacées

Les Fagales

Les Oléacées

Les Pinacées

Le platane