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Les profilines

samedi 21 août 2010, par Allerdata

La présence d’un autre allergène que Bet v 1 dans le pollen de bouleau a été suspectée dès 1983 par Ipsen au Danemark. Cette protéine IgE-réactive a été confirmée par Jarolim en 1989 et montrée correspondre à une profiline en 1991 par la même équipe à Vienne : c’était Bet v 2.

A la suite des observations de Calkhoven , il a été rapidement établi que des profilines IgE-réactives étaient présentes dans de nombreux pollens et aliments végétaux ; et qu’elles manifestaient une large réactivité croisée entre elles. La notion de « panallergène » a ainsi été introduite .

Ce « succès » des profilines est en grande partie du :

à la relative facilité d’obtention des protéines à partir des sources naturelles (solubilité, purification par chromatographie d’affinité)
à la confection dès le début des années 1990 de profilines recombinantes (ex. rBet v 2), lesquelles ont permis la détection de profilines cross-réactives dans de multiples produits.

En août 2010 la base Allerdata recensait pas moins de 102 profilines sur un total de 1533 protéines IgE-réactives :

49 dans des pollens
47 dans des aliments végétaux
3 dans d’autres produits végétaux : le latex d’hévéa, la feuille de tabac et Arabidopsis (une plante de laboratoire)
3 dans des acariens (D. pteronyssinus, Blomia et Suidasia) dont le statut de protéine IgE-réactive pourrait mériter d’être confirmé par d’autres études

L’ubiquité des profilines s’explique par leurs multiples fonctions dans les cellules eucaryotes : modifications du cytosquelette, signalisations, etc.. Elles ont, par exemple, un rôle important dans la germination du grain de pollen .

Les profilines sont de petites protéines (13-15 kDa), non glycosylées, et dont l’allergénicité est modérée :

d’un côté elles formeraient à l’état naturel des dimères ou plus , forme qui est suspectée favoriser l’allergénicité
mais d’un autres côté, étant des protéines ayant un homologue chez l’Homme, elles seraient moins immunogènes que des allergènes comme Bet v 1 (bouleau) ou Phl p 1 (fléole)

Réactivités croisées entre profilines

Les homologies entre profilines végétales sont élevées (70-85% d’identité séquentielle). Mais ces profilines ont une ressemblance assez modeste (30-40%) avec les profilines d’autres classes du vivant. Ainsi on n’a pas caractérisé de profiline fungique IgE-réactive jusqu’à présent.

Mais les profilines, comme d’autres protéines globulaires, ont avant tout des épitopes conformationnels . Ainsi :

une certaine homologie locale au niveau d’un épitope seulement peut permettre une réactivité croisée, même si elle est de faible affinité. C’est le cas pour la profiline humaine :
- Valenta avait noté quelques patients positifs pour la profiline humaine lors de son travail princeps sur Bet v 2
- Tordesillas a confirmé cette observation chez des patients allergiques au melon et positifs pour la profiline de melon Cuc m 2 : 10 de ces 17 patients étaient également positifs pour la profiline humaine
- la profiline humaine serait donc potentiellement un « auto-allergène »
à l’inverse, de fines différences au niveau des épitopes (y compris entre isoformes d’un même allergène ) peuvent susciter des discordances entre résultats pour telle ou telle profiline :
- si des concordances quasi-totales de positivité pour rPhl p 12 (fléole) et rBet v 2 (bouleau) ont été rapportées
- des écarts tantôt en faveur de Bet v 2 , tantôt en faveur de Phl p 12 sont possibles.
- de même, dans une une large étude utilisant le test ISAC, les positivités pour les 9 profilines du test allaient de 3,4% (rPhl p 12) à 7,2% (rMer a 1, mercuriale)
- et si les corrélations entre résultats chiffrés 2 à 2 sont bonnes , la pente de ces corrélations est souvent différente de 1, ce qui dénote de fines différences d’IgE-réactivité entre profilines.

On a donc d’une manière générale une large réactivité croisée, avec quelques variations : des épitopes communs + des épitopes plus ou moins spécifiques d’espèce .

En pratique, on retiendra qu’il n’est pas nécessaire de tester plusieurs profilines pour rechercher une sensibilisation profilinique chez un patient donné. Les puces type ISAC n’apportent pas de renseignement supplémentaire par rapport à un CAP isolé.

A l’heure actuelle on peut tester en CAP rBet v 2 (bouleau), rPhl p 12 (fléole), rPru p 4 (pêche) et rHev b 8 (latex). Ces deux derniers recombinants ont peu d’intérêt (cf. plus loin) et le choix se portera sur la profiline du pollen dominant de l’environnement du patient.

Quelles sont les prévalences d’IgE-réactivité des profilines ?

Les positivités pour une profiline sont fortement variables d’une étude à une autre, avec néanmoins un gradient Nord-Sud : beaucoup plus de patients sont réactifs aux profilines dans la partie méridionale de l’Europe.

Par ailleurs, les chiffres dépendant de la sélection des patients : les cohortes avec pollinose et allergie alimentaire ont des prévalences de positivité pour les profilines nettement plus fortes que les cohortes de polliniques sans allergie alimentaire. Les chiffres publiés vont ainsi de 2-5% à plus de 60% (ex. et voir aussi : Bet v 2).

La présence d’une IgE-réactivité profilinique reflète donc la présence d’un tableau allergique plus étendu :

allergie à des aliments végétaux en plus d’une pollinose
réactivité à plusieurs pollens plutôt qu’à un seul

Comme pour les polcalcines, on trouve avant tout des résultats positifs en profiline chez des patients présentant une multi-réactivité pollinique :

ces derniers étaient 64% parmi les sujets positifs en profiline, contre 12% en cas de profiline négative, dans une étude de l’équipe de Deviller
un test cutané positif pour rBet v 2 était noté chez 55% des patients positifs pour plusieurs pollens, mais chez aucun des mono-polliniques
une réactivité en profiline n’était vue que chez des patients positifs pour au moins 4 pollens, comme le montre la figure ci-dessous :

Le caractère un peu « accessoire » d’une sensibilisation aux profilines apparaît aussi dans la grande rareté des patients mono-profiline, c’est-à-dire positifs en profiline bien que négatifs pour les allergènes principaux et spécifiques du produit allergisant (ex. patient positif pour le seul pollen de bouleau, avec Bet v 2 positif avec Bet v 1 négatif) .

Ce qui pose la question de l’impact clinique d’une IgE-réactivité vis-à-vis des profilines : ces allergènes n’ont-ils qu’un intérêt diagnostique (= expliquer des tests cutanés ou in vitro discordants avec la clinique) ? Ou bien sont-ils aptes à participer à tout ou partie d’une réaction clinique chez certains patients ?

Ce point sera abordé ci-après. Mais il n’est pas vain de noter au passage que les remarques concernant la terminologie ont ici toute leur place : peut-on parler de patients « poly-sensibilisés » alors qu’il s’agit en grande partie d’un mirage, à savoir une (ou deux) vrai(s) sensibilisation(s) et de simples réactivités croisées positivant de nombreux autres produits … On voit l’intérêt de pouvoir confirmer la réalité d’une éventuelle multi-sensibilisation par le biais de tests orientés vers des allergènes spécifiques de ces autres produits.

Les profilines ont-elles un impact clinique ?

Profilines et pollinose

La rareté des cas de mono-réactivité à une profiline (cf. plus haut) ne favorise pas la réponse à la question de l’impact clinique des profilines au sein d’une allergie pollinique.

L’observation d’un patient présentant une rhinite en avril avec rBet v 1 négatif mais rBet v 2 positif constitue un cas assez rare : ici la pertinence clinique de Bet v 2 a été vérifiée en TC et à l’aide d’un TPN.

La profiline d’olivier, Ole e 2, a montré également son allergénicité dans une étude de patients Espagnols : 26% des polliniques à l’olivier avaient un TPN positif pour Ole e 2. Et des taux similaires étaient relevés avec d’autres profilines (armoise, Cynodon) .

Egalement en Espagne, il a été suggéré que la profiline du chénopode, Che a 2, jouait un rôle supérieur à celui de Che a 1, allergène spécifique de ce pollen .

Ces travaux rappellent que la réactivité vis-à-vis des profilines est plus fréquente en milieu méditerranéen : l’impact clinique pourrait de la sorte y apparaître plus nettement. En Italie, des scores cliniques plus sévères ont été notés chez des polliniques aux graminées s’ils étaient positifs en profiline .

On pourrait donc admettre que les profilines sont capables, dans certaines situations, de jouer un rôle additionnel dans la symptomatologie pollinique.

La question de l’efficacité d’une désensibilisation pollinique en présence d’une réactivité pour les profilines est abordée plus loin

Profilines et latex

Quelques études ont été réalisées : elles ne sont pas en faveur d’un rôle clinique notable de la profiline Hev b 8 dans l’allergie au latex (cf. l’article sur le latex).

Profilines et allergie alimentaire

Il est clair que de nombreux patients polliniques présentant une réactivité clinique pour des fruits et légumes sont IgE-réactifs en profilines . Mais l’opinion des experts est partagée :

pour van Ree et d’autres : impact clinique faible, voire nul
mais Asero ou Fernandez-Rivas accordent une importance non négligeable aux profilines

Ces opinions divergentes sont néanmoins du côté d’une faible allergénicité des profilines, comparativement aux LTP par exemple. De fait, l’expression clinique se résume le plus souvent à un syndrome oral.

Pourquoi des réactions plutôt locales ?

On attribue cette allergénicité modeste à l’instabilité des protéines :

si les profilines résistent en milieu salivaire
leur dégradation en milieu stomacal est quasi immédiate (< 1 min) . Les travaux montrant une certaine résistance digestive aux profilines sont exception .

Plus positifs sont les résultats de résistance thermique des profilines :

pour le céleri : profiline stable 30 min à 100°C , et 4 patients/6 encore positifs en TPODA après cuisson s’ils étaient Bet v 2 positifs, contre 1/5 si Bet v 2 négatifs
la profiline du melon résiste 15 min à 100°C et celle d’orange à la flash-pasteurisation des jus (30 sec à 98°C) .

Les profilines sont donc plus tables que les PR-10 (Bet v 1-like) et moins stables que les LTP. Malgré tout, à l’instar des observations concernant le soja, l’instabilité des profilines à la digestion doit être modulée : qu’en est-il en cas d’hypo-acidité gastrique ? Quel est l’effet de la matrice alimentaire , par exemple des pectines ?

Quels aliments avec impact clinique probable ?

Asero liste quelques aliments qui seraient assez caractéristiques d’une réactivité due aux profilines : les Cucurbitacées, la tomate et la banane surtout avec éventuellement aussi les agrumes et l’ananas .

Fernandez-Rivas y ajoute les fruits des Rosacées, dès lors qu’une réactivité LTP ou PR-10 est absente .

Ces opinions sont corroborées par des travaux sur le melon et sur les Rosacées en milieu méditerranéen.

Dans d’autres environnements, il faut évoquer le rôle suspecté des profilines dans l’association armoise – Apiacées (ex. céleri), ainsi que des observations relatives à des fruits exotiques (litchi , banane ou ananas ).

En cas de pollinose au bouleau il est difficile de distinguer la part de Bet v 2 de celle, majeure, de Bet v 1 (ex. pomme, noisette, Apiacées, kiwi, etc..). Et la pollinose aux graminées ne semble pas susciter d’allergie alimentaire aux céréales .

Une discussion plus large des différentes composantes possibles dans les réactions alimentaires à des produits végétaux est abordée dans d’autres chapitres. On pourra s’y référer :

melon et Cucurbitacées
ananas, litchi et aliments latex
pêche et Rosacées, ainsi que pour la démarche diagnostique

Positivité pour un panallergène (profiline, polcalcine) et immuno-thérapie

Certains auteurs ont soutenu la thèse d’une mauvaise indication pour une désensibilisation pollinique en cas de positivité pour les profilines et/ou les polcalcines , sauf peut-être en zone méditerranéenne (cf. Les graminées et Le bouleau).

Il n’a pas été fait d’étude prospective avalisant cette position … qui pourtant concerne une décision thérapeutique très courante.

En quoi une réactivité pour les profilines et/ou les polcalcines pourrait, en soi, perturber l’efficacité d’une immuno-thérapie (IT) ? Divers arguments vont plutôt dans l’autre sens :

les extraits utilisés en IT contiennent-ils ces protéines ? La réponse est souvent négative
s’ils en contiennent, est-ce gênant ? Non si l’on se réfère au faible potentiel allergénique des profilines et des polcalcines
et, si cela était le cas, en quoi cela serait-il dommageable à l’efficacité de l’IT ? On pourrait même espérer une action sur un syndrome oral induit par la pollinose …

La motivation qui sous-tend la prise en compte des panallergènes dans l’efficacité de l’IT semble plutôt résulter d’une constatation courante : quand un patient est positif pour une profiline (et plus encore pour une polcalcine), il a de grandes chances de présenter une multi-réactivité pollinique en TC ou in vitro.

Aussi, si la décision pour une IT ne se base que sur des tests positifs pour des extraits de pollens, il est clair que c’est en dépit du bon sens : il est probable que la réalité clinique est beaucoup plus restreinte, avec par exemple 1 seul pollen cliniquement pertinent et la positivité pour les autres (y compris en TC) n’étant qu’une réactivité croisée par le biais de profilines et/ou polcalcines.

Ce n’est donc pas la présence ou l’absence d’une IgE-réactivité pour une profiline ou pour une polcalcine qui importe : c’est la confirmation que le patient s’est bien sensibilisé vis-à-vis de tel ou tel pollen parmi ceux trouvés positifs avec des extraits en TC ou in vitro.

Et il suffit pour cela d’utiliser la batterie des allergènes purs (recombinants ou non) et a priori spécifiques de tel pollen (ou groupe botanique). Si on ne possède pas un outil pour toutes les sortes de pollens à l’heure présente, la plupart des situations cliniques peuvent être abordées avec :

rBet v 1 : bouleau et autres Fagales
rPhl p 1+5 (et parfois rPhl p 2) : graminées tempérées (fourragères et céréalières)
rPar j 2 : pariétaires
nArt v 1 : armoise
nAmb a 1 : ambroisies
nCup a 1 : Cupressacées
nOle e 1 : Oléacées (dont frêne)
nSal k 1 : soude (Amaranthacée)

Dès lors que l’anamnèse et les données cliniques coïncident avec la positivité pour un allergène spécifique de tel pollen, on comprend mal pourquoi l’indication d’IT serait affaiblie par la présence d’une réactivité pour une profiline ou une polcalcine.

La question paraît donc se résumer à la seule limite d’un nombre raisonnable d’extraits :

si le patient est effectivement positif pour 3 ou 4 allergènes spécifiques (= 3 ou 4 pollens différents), est-il cliniquement symptomatique à toutes les périodes de pollinisation de ces pollens ? Dans la négative, l’IT sera guidée par la clinique.
si le patient est symptomatique à différentes périodes de l’année, le choix sera effectivement plus difficile. On pourra cependant intégrer dans cette expression clinique l’éventuelle intervention de causes plus ou moins perannuelles (ex. acariens, phanères, ficus, voire parfois des pollens comme les pariétaires).

L’abord des poly-réactivités polliniques n’est pas toujours simple. Ainsi, dans une étude portant sur 200 patients Milanais et intégrant des TC pour 9 pollens différents ainsi qu’une profiline et un polcalcine, les auteurs ont trouvé 12 cas où au moins 6 pollens étaient positifs bien que profiline et polcalcine soient négatifs . Or il y avait autant de patients se déclarant gênés sur une seule période de l’année (printemps) que de patients qui, plus logiquement, évoquaient une symptomatologie sur les 3 périodes de l’année (arbres, graminées, herbacées).

Il aurait fallu, dans cette étude, mieux discriminer les véritables sensibilisations chez ces patients multi-réactifs en TC en étudiant leurs réactivités pour des allergènes précis (ex. rBet v 1, rPhl p 1, nAmb a 1, etc..). Ici, comme souvent rencontré ailleurs, le terme de « poly-sensibilisation » n’est pas fondé faute de preuves au niveau moléculaire.

Les deux exemples suivants montrent l’application possible d’une démarche moléculaire, étant entendu que la réponse à ces tests n’a pas pour objectif d’avaliser ou de rejeter le bien-fondé d’une immuno-thérapie mais de clarifier dans un premier temps le tableau d’une multi-positivité pollinique :

un patient Espagnol présentait des TC positifs de nombreux pollens et pour une polcalcine (9 mm) et était négatif en profiline . Voici ci-dessous ses résultats :

	TC (mm)	IgE (kU/l)
armoise	4	Art v 1 0,27
cyprès	4,5	Cup s 1 <0,10
olivier	13	Ole e 1 1,80
pariétaire	8	Par j 1 <0,10
graminées	8	Phl p 5 10,9
plantain	7	Pla l 1 <0,10

Chez ce patient, seuls les pollens d’olivier et de graminées avaient une pertinence clinique.

une patiente Autrichienne, pollinique aux graminées, mais dont les TC pour le bouleau, l’olivier, l’armoise, l’ambroisie et la pariétaire étaient positivés par une réactivité vis-à-vis des polcalcines : in vitro rPhl p 7 était positif mais rBet v 1 et rBet v 2 étaient négatifs. Résultats qui ont été confirmés en TC (cf. la photo des TC en fig.4 de ce travail ).

Il est possible de tester en CAP un mélange rPhl p 7 + rPhl p 12. Mais il est relativement fréquent que les réactivités pour les profilines et pour les polcalcines soient dissociées chez le même patient : il semble donc plus judicieux de tester séparément une profiline (rPhl p 12 ou rBet v 2) et une polcalcine (rPhl p 7 ou rBet v 4), le choix étant guidé par le pollen dominant de l’environnement du patient (graminées ou bouleau).

Que la réponse soit positive ou négative pour ces 2 types de panallergènes, cela n’évitera pas de devoir préciser les sensibilisations pertinentes par la recherche d’une réactivité pour un allergène plus spécifique de chacun des pollens auxquels le patient s’avère positif. En effet, c’est cette recherche qui justifie le choix et le bien-fondé de l’IT plus que la positivité ou la négativité pour une profiline ou une polcalcine.

[1] - Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O et al. IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa. Allergy 1989;44:385-395

IgE and IgG antibody responses to birch pollen antigens were studied by means of immunoblotting experiments testing 58 sera from patients with Type I allergy to birch pollen. 56/58 patients showed IgE antibodies reactive with Bet v I, a 17 kilodalton (kD) pollen protein. 2D-electrophoresis/immunoblot revealed a heterogeneity of that protein. Ten spots (pH 4.9–5.9) could be detected, presumably representing differentially glycosylated isoallergens. In 33/58 patients, there was no evidence of IgE antibodies directed against allergens other than Bet v I. However, in 25/58 of patients' sera, 11 minor allergens (13, 15, 18, 27, 29, 32, 36, 39, 44, 57, and 68 kD) with individual incidences from 1.7% to 17.2%, were identified. All proteins were also recognized by the patients' IgG antibodies: in the case of Bet v I recognition was weak, whereas the IgG response to the minor allergens was pronounced. Sera from healthy individuals showed similar IgG antibody responses, but no IgG to the 15, 27, and 29 kD proteins. Our results suggest that IgG directed against minor allergens may function as trapping antibodies in healthy individuals. Too low or lacking amounts of anti-Bet v I IgG may facilitate an allergic reaction.

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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] - Calkhoven PG, Aalbers M, Koshte VL, Pos O, Oei HD, Aalberse RC. Cross-reactivity among birch pollen, vegetables and fruits as detected by IgE antibodies is due to at least three distinct cross-reactive structures. Allergy 1987;42:382-390

Sera of patients suffering from birch pollinosis were studied in the radio-allergo-sorbent test (RAST) for the presence of IgE antibodies to various allergens of vegetable origin. The sera selected were positive in the RAST for both birch pollen and fruits. IgE antibodies directed against at least three different cross-reacting determinants in birch pollen were detected. In addition to periodate-susceptible cross-reacting determinants, which are found on a number of glycoproteins, two non-related periodate-resistant determinants were found in birch pollen, with molecular weights of 20 and 18 kD, respectively. The 20-kD component appears to be responsible for the co-occurrence of the binding of IgE to allergens of fresh fruits, whereas the 18-kD component appears to cause the cross-reactivity among grass pollen, potato and fruits.

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[4] - Vallier P, Dechamp C, Valenta R, Vial O, Deviller P. Purification and characterization of an allergen from celery immunochemically related to an allergen present in several other plant species. Identification as a profilin. Clin Exp Allergy 1992;22:774-782

The purification of a 15 kD allergen from celery was obtained by a four step procedure. Evidence for at least two isoallergenic forms was obtained after analysis by two-dimensional-electrophoresis. A rabbit polyclonal antibody raised against this purified allergen allowed us to confirm our precedent results on the occurrence of allergenically and molecular mass-related components in celery and birch and mugwort pollens. In addition such components were also present in numerous other species like Cynodon dactylon, Sorghum halopense, Poa pratensis, Ambrosia elatior and in apple and carrot. The 15 kD allergen was identified as profilin by use of a specific rabbit polyclonal antibody that recognized a recombinant birch profilin. In addition, the purified celery allergen binds IgE from sera of patients allergic to birch profilin. These results reinforce the concept of profilin as a panallergen responsible in some patients for cross-allergic manifestations to various and unrelated species of grasses, weeds, trees, vegetables and fruits

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[5] - 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

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[6] - Lai FY, Loo AHB, Ramjan SFR, Reginald K, Shang HS, Chew FT. Pan-allergenicity of profilins - tests using recombinant protein homologues. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°581

Profilins are important pan-allergens and play important roles in cross-reactivity in pollinosis, oral and latex allergy syndrome. Current assumptions on cross-reactivity are based mainly on total proteins or comparisons among few recombinant protein homologues. To test if there are phyletic boundaries in profilin pan-allergenicity, orthologous profilin proteins from diverse plant species : Capsicum annuum- Solanaceae, Daucus carota-Apiaceae, Elaeis guineensis-Arecaceae, Hevea brasiliensis- Euphorbiaceae) as well as a storage dustmite species(Suidasia medanensis - Acaridae) were tested for patterns of reactivity in IgE binding.7 profilin orthologues and paralogous isoforms in D. carota and H. brasiliensis were isolated (RT-PCR) and recombinant proteins expressed as fusion-tag proteins in expression vector (pet32a+). Immunodot-blots were carried out using sera of 10 latex postive and one non-atopic sera. Results:7/10 atopic sera showed positive IgE binding and interestingly, all positive reacting sera were to more than one recombinant protein, suggesting cross-reactivity. All positive reacting sera showed IgE binding to three recombinant homologues (a D. carota isoform, a H. brasiliensis isoform and C. annuum). All postive reactants were reacting to only one H. brasiliensis isoform. The two isoforms differ significantly with 9% amino acid differences. 6 reacted to one profilin isoform in D. carota and 4 of these to both isoforms. The D. carota isoforms differ slightly (2%) in terms of amino acid differences. One sera in particular had postive reactions to all recombinants (even dustmite profilin) except to one isoform of D. carota and another in H. brasiliensis. Although this patient had a reaction to one isoform of H. brasiliensis and to E. guineensis, the sera did not react to the H. brasiliensis that was phylogenetically closer to E. guineensis. The results show differences in IgE reactivity to isoforms of profilins even if they originate from the same organism, making inferences at cross-reactivity more complex. A purely phylogenetic explanation for cross-reactivity may be unfounded. Cross-inhibition tests are needed to to see if the patterns of reactivity to several homologues from each sera are based on cross-reactions to profilin homologues from diverse phyla.

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[7] - Angus AC, Ong ST, Chew FT. Sequence tag catalogs of dust mite-expressed genomes: utility in allergen and acarologic studies. Am J Pharmacogenomics 2004;4:357-369

Dust mites are a major source of indoor allergens. They contain a large number of components that react with immunoglobulin (Ig) E in individuals with allergies and are capable of inducing sensitization, and allergic respiratory and cutaneous diseases. With a significant proportion of the population affected in some way by mite allergies, it is essential that we improve our understanding of these organisms so that control strategies could be defined and its allergens better understood. Thus, we have initiated a project using the expressed sequence tagging (EST) strategy to study the major species of dust mites associated with allergic diseases, in particular, the American house dust mite, Dermatophagoides farinae, as well as Blomia tropicalis, the most prevalent mite in domestic tropical dwellings. The work has recently been expanded to include 'storage' mites such as Tyrophagus putrescentiae, Acarus siro, Lepidoglyphus destructor, Glycyphagus domesticus, Suidasia medanensis, and Aleuroglyphus ovatus. More than 50% of the initial 3000 ESTs from the D. farinae and B. tropicalis dust mites showed significant matches to known genes and were categorized into eight functional groups (such as proteins involved in metabolism, gene expression, protein synthesis, cell signaling, etc.). Of specific interest, however, were the homologs to known mite allergens, in addition to a number of sequences bearing significant homology to allergens from non-mite sources previously not known to exist in mites. The availability of these allergen sequences has facilitated their expression and subsequent characterization in our laboratory in terms of their IgE-binding reactivity. The wealth of sequence information, generated via the EST project, has also facilitated the identification of polymorphic forms of allergens, the investigation of differential gene expression under various environmental conditions via DNA microarrays, as well as the analysis of protein level expression profiling via the proteomics approach. Additionally, ESTs have also ameliorated the understanding of the phylogenetic relationships between mites, and enabled the isolation of gene products crucial for life processes so that mite control strategies can be more effectively devised. Taken together, the utilization of the EST strategy has opened up numerous new avenues by which the allergist can engage more effectively in the study of dust mites with the ultimate aim of developing appropriate treatment regimens for mite-induced allergy.

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[8] - Morales S, Jiménez-López JC, Castro AJ, Rodríguez-García MI, Alché JD. Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination. J Microsc 2008;231:332-341

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the 'effective' apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization-enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube. The results shown here could be interpreted as signs of a massive dissociation of the actin-profilin complexes, mobilization of actin monomers, and therefore, an intense activity of the actin cytoskeleton. The extensive release of allergenic proteins from the pollen grain into the surrounding aqueous media, as described here for profilin, may help us to understand the mechanisms by which these allergens might come in contact with the human mucosa, therefore triggering the symptoms of allergy.

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[9] - Willerroider M, Fuchs H, Ballmer-Weber BK, Focke M, Susani M, Thalhamer J, et al. Cloning and Molecular and Immunological Characterisation of Two New Food Allergens, Cap a 2 and Lyc e 1, Profilins from Bell Pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum). Int Arch Allergy Immunol 2003;131:245-255

BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food . OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins . METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA . RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin . CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.

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[10] - 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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[11] - Radauer C, Breiteneder H. Pollen allergens are restricted to few protein families and show distinct patterns of species distribution. J Allergy Clin Immunol 2006;117:141-147

BACKGROUND: Inhalative allergies are elicited predominantly by pollen of various plant species. However, a classification of the large number of identified pollen allergens is still missing . OBJECTIVE: To analyze pollen allergen sequences with respect to protein family membership, taxonomic distribution of protein families, and interspecies variability . METHODS: Protein family memberships of all plant allergen sequences from the Allergome database were determined by using the Protein Families Database of Alignments and Hidden Markov Models. The taxonomic distribution of pollen allergens was established from the Integrated Taxonomic Information System. Members of abundant pollen allergen families were compared with allergenic and nonallergenic homologues by database similarity searches and multiple sequence alignments . RESULTS: Pollen allergens were classified into 29 of 7868 protein families. Expansins, profilins, and calcium-binding proteins constitute the major pollen allergen families, whereas most plant food allergens belong to the prolamin, cupin, or profilin families. Pollen allergens were revealed to be ubiquitous (eg, profilins), present in certain plant families (eg, pectate lyases), or limited to a single taxon (eg, thaumatin-like proteins). Allergenic plant profilins constitute a highly conserved family with sequence identities of 70% to 85% among each other but low identities of 30% to 40% with nonallergenic profilins from other eukaryotes, including human beings. Similarly, allergenic polcalcins possess sequence identities of 64% to 92% but show low identities of 39% to 42% to related nonallergenic calmodulins and calmodulin-like proteins from vegetative plant tissues and man . CONCLUSION: This classification of pollen allergens into protein families will aid in predicting cross-reactivity, designing comprehensive diagnostic devices, and assessing the allergenic potential of novel proteins.

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[12] - Radauer C, Willerroider M, Fuchs H, Hoffmann-Sommergruber K, Thalhamer J, Ferreira F et al. Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis. Clin Exp Allergy 2006;36:920-929

Background: Profilins are cross-reactive plant allergens responsible for multiple pollen sensitisation and pollen-associated food allergy. It is assumed that profilins from different species are immunologically equivalent and can be represented by a single profilin in diagnostic assays. However, some studies suggest partial or even lacking IgE cross-reactivity between certain profilins. We aimed to obtain a quantitative assessment of the contributions of conserved and species-specific epitopes to IgE binding of plant profilins. Methods: We compared model structures of profilins from timothy and mugwort pollen, celery tuber and bell pepper with the structures of birch pollen and latex profilins. Based on these structures, we predicted potential conformational epitopes that consisted of contiguous patches of at least 20% surface-exposed residues. Celery tuber and timothy pollen profilins were purified from their natural sources, and profilins from birch and mugwort pollen, bell pepper and latex were expressed in E. coli. Structural integrity of all purified proteins was confirmed by circular dichroism spectroscopy. IgE ELISAs and ELISA inhibitions using these profilins and sera from 22 profilin-sensitised allergic patients were carried out. Results: Peptide backbone conformations of all six profilins were highly similar. Epitope prediction yielded 11 overlapping potential epitopes. Nine of them were classified variable, two contained high proportions of conserved residues. IgE from all sera bound to all tested profilins and the amounts of IgE binding to different profilins were highly correlated. However, IgE inhibition experiments revealed that up to 60% of total IgE binding was mediated by species-specific epitopes. The extent of cross-reactivity among profilins from timothy, birch, latex and celery was greater than to mugwort and bell pepper profilins. This pattern fitted the pattern of sequence similarities among one of the predicted variable epitopes. Patients whose IgE bound to crossreactive epitopes on profilins displayed allergic reactions to a greater number of plant foods compared with patients containing IgE directed to speciesspecific epitopes. Conclusion: The large extent of cross-reactivity among plant profilins justifies the use of a single profilin in diagnostic assays. However, a considerable amount of IgE bound to variable epitopes, which was confirmed by ELISA inhibition and by in silico epitope prediction.

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[13] - Rihs HP, Chen Z, Rueff F, Petersen A, Rozynek P, Heimann H, et al. IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes. J Allergy Clin Immunol 1999;104:1293-1301

Soybean proteins are constituents of a number of food products and represent a panel of potential allergens. Thus far, little is known about the molecular characteristics of soybean allergens. OBJECTIVE: The aim of this study was to identify the soybean profilin by PCR-based complementary (c)DNA cloning and to elucidate its allergenic characteristics. METHODS: Highly degenerate profilin-specific primers were used to identify, by means of PCR, 2 soybean profilin isoforms (GmPRO1 and GmPRO2) by using soybean cDNA as a target. One isoform (GmPRO1) with a length of 394 bp corresponding to 131 amino acid residues was subcloned and expressed in fusion with the maltose-binding protein. Moreover, 3 overlapping recombinant soybean profilin fragments comprising amino acid residues 1-65, 38-88, and 50-131 were also prepared as maltose-binding protein fusion proteins. IgE-binding reactivity of the recombinant proteins and the cross-reactivity of soybean profilin with birch profilin was studied by immunoblotting, enzyme-linked allergosorbent assays (EASTs), and competitive inhibition experiments by using serum samples from 13 soybean-sensitized subjects. RESULTS: Results of immunoblot analysis, EAST, and EAST-inhibition experiments indicate the presence of profilin in soybean extract. The recombinant soybean profilin (rGly m 3) was recognized by IgE in 9 (69%) of the 13 sera tested. Only the full-length rGly m 3 was able to bind with IgE antibodies, whereas the 3 soybean profilin fragments did not show significant binding reactivity, indicating that the IgE binding to rGly m 3 depends on the integrity of a conformational structure, which was not present in the overlapping profilin fragments. The rGly m 3 cross-reacted with birch pollen profilin (Bet v 2), and the IgE binding to Bet v 2 could be inhibited by rGly m 3. CONCLUSIONS: rGly m 3 represents a new soybean allergen with well-characterized primary sequence, and its IgE-binding reactivity is mediated by conformational epitopes.

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[14] - 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

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[15] - Tordesillas L, Pacios LF, Palacín A, Cuesta-Herranz J, Madero M, Díaz-Perales A. Characterization of IgE epitopes of Cuc m 2, the major melon allergen, and their role in cross-reactivity with pollen profilins. Clin Exp Allergy 2010;40:174-181

BACKGROUND: Plant profilins are described as minor allergens, although with some exceptions in foods such as melon, watermelon or orange. In fact, they could be responsible for many cross-reactions among distantly related species. This is likely to be a consequence of the presence of common epitopes. OBJECTIVE: To characterize the B epitopes of Cuc m 2, a model of plant food profilin, using phage display techniques and to compare with other profilins, such as those of timothy grass and birch pollen, and human I profilin, to understand the mechanism of cross-reaction among members of this family. METHODS: IgE of melon-allergic patients was used to select clones from a phage display 12 mer peptide library. After two rounds of screening, Cuc m 2-specific clones were eluted and the DNA insertion sequenced. The residues of each clone were mapped on the Cuc m 2 surface to define a mimotope, which was also localized on the three-dimensional surfaces of other profilins. RESULTS: Seventeen melon-allergic patients were selected. Sera from each of them recognized the melon profilin, Cuc m 2, but the majority also recognized Phl p 12 or Bet v 2, timothy grass-, and birch-pollen profilins, respectively. A Cuc m 2 mimotope was defined and mapped onto its surface giving the following sequence: S(2)W(3)A(5)Y(6)D(9)H(10)T(111)P(112)G(113)Q(114)N(116)M(117)R(121)L(122). The homologous residues in Phl p 12 and Bet v 2 had almost identical sequences. By contrast, the homologous sequence in human profilin showed many differences. CONCLUSIONS: The identified mimotope could be involved in cross-reactions among food and pollen profilins. Many of these cross-reactions observed in the clinical realm could be explained by the presence of a common epitope found in food and pollen allergens. A new strategy of immunotherapy based on this IgE region could be used in alternative immunotherapy strategies.

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[16] - Lai FY, Loo AHB, Ramjan SFR, Reginald K, Shang HS, Chew FT. Pan-allergenicity of profilins - tests using recombinant protein homologues. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°581

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

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

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[18] - 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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[19] - 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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[20] - Lidholm J, Marknell DeWitt A, Mattsson L, Ostling J, Lilja G, Nordlund M, et al. An Objective Approach for the Assessment of Interchangeability of Cross-Reactive Food Allergens in Diagnostic Tests for Specific IgE. J Allergy Clin Immunol 2007;119(1 suppl):S194

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[21] - 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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[22] - Scala E, Alessandri C, Bernardi ML, Ferrara R, Palazzo P, Pomponi D et al. Cross-sectional survey on immunoglobulin E reactivity in 23 077 subjects using an allergenic molecule-based microarray detection system. Clin Exp Allergy 2010;40:911-921

Summary Background The availability of allergenic molecules and high-throughput microtechnologies allow the collection of a large number of IgE results at the same time in a single test. This can be carried out applying the test in the routine diagnostic work-up. Objective The aim of this study was to make a cross-sectional evaluation of the raw prevalence of IgE reactivity to allergenic molecules in serum samples from a cohort of Italian patients using an innovative tool. Methods The ISAC, a microarray system, has been used for specific IgE detection using 75 different allergenic molecules. Sera were collected from 23 077 unselected consecutive individuals complaining about any allergic disease. Results Sixteen thousand four hundred and eight of 23 077 patients had IgE to at least one of 75 allergenic molecules. The top-ranked molecules in this cohort were Cup a 1 (42.7%), Der f 2 (38.7%), and Phl p 1 (37.9%), whereas all the other allergens tested scored in a range between 36.8% and 0.04%, including the first food allergen, Pru p 3, ranked 15th (9.79%). Prevalence varied quite markedly depending on the age range considered, and showing a different behaviour in the lifetime sensitization process. Unsupervised two-way hierarchical clustering analysis generated distinctive patterns of reactivity as the result of IgE recognition of either homologous allergens belonging to different biological sources or non-homologous belonging to the same biological source. Conclusions Allergen-based microarray is a tool for the detection of IgE-related sensitization to panels of allergens and gives a more precise and comprehensive evaluation for an IgE-based epidemiology. This insight brings data for better understanding of the sensitization process.

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[23] - Radauer C, Willerroider M, Fuchs H, Hoffmann-Sommergruber K, Thalhamer J, Ferreira F et al. Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis. Clin Exp Allergy 2006;36:920-929

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[24] - Elfman L, Svensson M, Lidholm J, Pauli G, Valenta R. Different profiles in specific IgE to rBet v 1 and rBet v 2 in patients allergic to birch pollen from six countries. Int Arch Allergy Immunol 1997;113:249-251

There are differences in IgE reactivity to rBet v 1 and rBet v 2 among allergic patients from the six countries studied. The complexity of the reactivity profile tends to be greater in individuals from the central/southern parts of Europe compared to Sweden and Finland. There is a good agreement between in vitro diagnosis with Pharmacia CAP System and Skin prick testing when using the same reagents, rBet v 1 and rBet v 2.

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[25] - Scheurer S, Wangorsch A, Haustein D, Vieths S. Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2. Clin Exp Allergy 2000;30:962-971

Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. OBJECTIVES: Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. METHODS: Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. RESULTS: The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. CONCLUSIONS: Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses

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[26] - Rihs HP, Chen Z, Rueff F, Petersen A, Rozynek P, Heimann H, et al. IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes. J Allergy Clin Immunol 1999;104:1293-1301

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[27] - Petersen A, Vieths S, Aulepp H, Schlaak M, Becker WM. Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens. J Allergy Clin Immunol 1996;98:805-815

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.

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[28] - van Ree R, Fernández-Rivas M, Cuevas M, van Wijngaarden M, Aalberse RC. Pollen-related allergy to peach and apple: an important role for profilin. J Allergy Clin Immunol 1995;95:726-734

Birch pollinosis is often accompanied by allergy to fruits such as peach and apple. Bet v I is of major importance as cross-reactive allergen for this combined allergy. We studied a group of patients with combined grass pollinosis and fruit allergy from an area virtually without birch trees. OBJECTIVE: The aim of this study was to investigate the possible involvement of profilin and carbohydrate groups as cross-reactive structures in pollen and fruits. METHODS: RAST inhibition was performed to measure cross-reactive IgE to pollen and fruits. The presence of IgE against profilin was determined in a RAST with purified grass profilin, and IgE against carbohydrate structures was determined in a RAST with proteinase K-digested grass pollen extract. The biologic activity of IgE in response to profilin was tested by in vitro histamine release and skin prick tests. RESULTS: IgE against fruits was shown to be largely cross-reactive with grass pollen. The majority of the patients had IgE against profilin (12 of 16) and carbohydrate structures (9 of 10). Profilin was shown to have biologic activity, in both histamine release and skin prick tests. CONCLUSION: Profilin is an important allergen for patients with combined grass pollen/fruit allergy in areas without birch trees.

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[29] - Fernández-Rivas M, Van Ree R, Cuevas M. Allergy to Rosaceae fruits without related pollinosis. J Allergy Clin Immunol 1997;100:728-733

BACKGROUND: Rosaceae fruit allergy is frequently associated with birch pollinosis in Central and Northern Europe and with grass pollen allergy in Central Spain. The main cross-reactive structures involved for birch pollinosis are Bet v 1 and profilin, and for grass pollinosis they are profilin and carbohydrate determinants. Rosaceae fruit allergy can occasionally be observed in patients without pollinosis . OBJECTIVE: We investigated the clinical presentation and the allergens involved in allergy to Rosaceae fruit without pollinosis . METHODS: Eleven patients from Central Spain allergic to apples, peaches, and/or pears but not to pollens were compared with 22 control subjects with combined grass pollen and fruit allergy. Skin prick tests and RASTs to apple, peach, and pear were performed. Cross-allergenicity was studied by RAST inhibition. Bet v 1 was tested with an indirect RAST, and profilin was tested in skin prick tests, histamine release, and RAST . RESULTS: Rosaceae fruit allergy without pollinosis is severe with 82% of patients reporting systemic symptoms, mainly anaphylaxis (73%), whereas oral symptoms are less frequent (64%). Anaphylactic shock was observed in 36% of patients. The fruit allergens involved showed cross-reactivity among Rosaceae species but were not related to profilin or Bet v 1. Ninety-one percent of patients with combined grass pollinosis and fruit allergy reported oral allergy, 45% reported systemic symptoms, 18% reported anaphylaxis, and 9% reported anaphylactic shock . CONCLUSION: Allergy to Rosaceae fruits in patients without a related pollen allergy is a severe clinical entity. Profilin- and Bet v 1-related structures are not involved in Rosaceae fruit allergy without pollinosis.

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[30] - 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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[31] - 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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[32] - 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

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[33] - Dubost R, Ruet N, Deviller P. Incidence de la sensibilisation à la profiline dans une population allergique aux pollens: responsabilité de la profiline dans des polysensibilisations polliniques chez des patients à taux normal d'IgE totales. Allerg Immunol (Paris) 2000;32:199-206

The study had the aim of establishing the incidence of sensitization to profilin (a panallergen found in pollens and foods of vegetal origin) in pollen-allergic patients. We evaluated the consequences of such sensitizations on the results of specific IgE, the positivity of skin tests and clinical signs. METHODS: 94 consenting patients, allergic to pollens (trees and/or grasses and/or weeds) replied to a questionnaire and had skin tests to purified profilin and measurement of serum anti-profilin IgE. RESULTS: Two groups were defined: one group was sensitized to profilin (GSP), with positive skin test and anti profilin IgE of 31 patients, and a group non-sensitive to profilin (GNSP) (negative skin test and anti-profilin IgE) of 41 patients. Discordant results were found in 22 patients. Taking in account the two groups, sensitization to profilin was 43%. The two groups were homogenous for age, sex, ethnics and clinical signs. Food allergy was more frequent but not statistically different (p = 0.09) in the group GSP (51.6%) than the GNSP (31.7%), in particular allergy to fruits of the Rosaceae family. Pollen polysensitization (to three species, trees, weeds and grasses) was more frequent in the GSP group (64.5%) than the GNSP (12.5). Polysensitization to pollens and foods was also more frequent in the sensitized group (65.5%) than in the non-sensitized group (12.5%). In a sub-group with normal levels of total IgE pollen polysensitization was more frequent in patients who were sensitive to profilin. On biological investigation, sensitization to profilin influenced the result of anti-latex IgE and also the IgE to many vegetal allergens. These results show the value of seeking a sensitization to profilin in patients with pollinoses.

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[34] - Mari A. Multiple pollen sensitization: a molecular approach to the diagnosis. Int Arch Allergy Immunol 2001;125:57-65

BACKGROUND: Sensitization to multiple pollen species is a frequent diagnostic event. Several allergenic molecules with a high level of homology have been identified in divergent pollen families and named panallergens. METHODS: We sought to define the criteria to evaluate the prevalence of the multiple pollen sensitization, to identify specific markers of this condition, and to correlate them with the underlying allergic disease. Patients presenting an allergic respiratory disease underwent skin testing with 23 pollens. Patients fulfilling predefined selection criteria were grouped and classified as having multiple pollen sensitization. Patients in each subgroup were tested for IgE to rBet v 2, rJun o 2, rBet v 1, rPhl p 5 and bromelain. Demographical, allergological and clinical data were recorded in the subgroup of patients with multiple pollen sensitization. RESULTS: Seventeen percent of the pollen-sensitized patients formed the multiple pollen-sensitized subgroup. These subjects were positive for most of the pollen species tested regardless of known exposure to them. None of the subjects sensitized to less than six pollen species were positive to panallergens, whereas 55% of the sera of the multiple pollen-sensitized group were positive to rBet v 2, and 15% to rJun o 2. IgE to rBet v 1 and rPhl p 5 were found positive in all the subgroups. Age, gender, bronchial asthma, oral allergy syndrome, skin test reactivity and previous specific immunotherapy differed significantly when these two subsets were considered. CONCLUSIONS: Allergy diagnosis based on allergenic molecules is crucial in the patient with multiple pollen sensitization. This condition appears to be determined by the sensitization to defined allergenic components (panallergens) rather than by pollen of multiple species as such. Detection of IgE to nonpanallergenic molecules allows to identify more relevant allergenic sources. Clinical aspects of the underlying allergic disease (e.g. asthma and oral allergy syndrome) seem to be differently related to IgE reactivity to panallergens

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[35] - Asero R, Jimeno L, Barber D. Preliminary Results of a Skin Prick Test–Based Study of the Prevalence and Clinical Impact of Hypersensitivity to Pollen Panallergens (Polcalcin and Profilin. J Investig Allergol Clin Immunol 2010;20:35-38

Background: Calcium-binding proteins (polcalcins) and profi lin are cross-reacting panallergens that sensitize a minority of pollen-allergic patients. Their clinical relevance remains controversial. Objective: To assess the clinical relevance of hypersensitivity to polcalcin and profi lin detected by skin prick test (SPT) in a large group of pollen-allergic patients. Methods: Two hundred pollen-allergic adults (101 men, 99 women; mean age 34 years) underwent SPT with 9 pollens present in the geographical area of the study. Hypersensitivity to panallergens was detected by SPT with date palm polcalcin and profi lin. Allergy to birch and/or cypress, grass and/or pellitory, and ragweed and/or mugwort were associated with 3 symptomatic periods, respectively, late February to mid-May, late April to mid-July, and mid-August to late September. Results: Sixteen (8%) patients reacted to date palm polcalcin; 7/7 (100%) corecognized the grass polcalcin Phl p 7 in vitro. Clinically, only 4 (25%) had symptoms in all 3 seasonal periods. Forty (20%) patients reacted to profi lin; only 32 (80%) reacted to cypress, and 22 (55%) to pellitory. Only 4 (10%) patients had symptoms during all 3 seasonal periods. Six patients (3%) were cosensitized to both polcalcin and profi lin. Conclusions: The clinical relevance of hypersensitivity to pollen panallergens is often limited; many allergic patients have symptoms only during the central period, suggesting primary grass sensitization. Profi lin-allergic patients often do not corecognize pellitory and cypress pollen. In vivo component-resolved diagnosis of seasonal respiratory allergies is a promising approach that might lead to cost reduction and a faster defi nition of pollen-allergic cases.

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[36] - 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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[37] - 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

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[38] - 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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[39] - Martínez A, Asturias JA, Monteseirín J, Moreno V, García-Cubillana A, Hernández M, et al. The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen. Allergy 2002;57(suppl. 71):17-23

Many works have dealt with the study of the allergenic relevance of profilin from allergenic extracts, mainly derived from pollens and vegetable foods. Olive pollen extracts also contain a profilin allergen (Ole e 2). This protein has been characterized in detail, so the aminoacid sequence of three isoforms and the structural model of one of them are already known. The prevalence of Ole e 2 for olive allergenic patients has been evaluated by different in vivo and in vitro methods, and the results compared with those obtained for another pollen profilins.

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[40] - Barderas R, Villalba M, Pascual CY, Batenero E, Rodriguez E. Profilin (Che a 2) and polcalcin (Che a 3) are relevant allergens of Chenopodium album pollen: Isolation, amino acid sequences, and immunologic properties. J Allergy Clin Immunol 2004;113:1192-1198

Background Little is known about the molecular properties of chenopod allergens. Recently, profilin and 2 EF-hand calcium-binding protein (polcalcin) have been shown to play a role in chenopod pollinosis. Objective : We sought to analyze these panallergens in chenopod pollen and to evaluate their involvement in the allergy to this biologic source. Method s : Profilin and polcalcin were purified to homogeneity and characterized by using spectrometric and chemical methods. Immunologic analyses were performed by means of immunoblotting, ELISA, and competitive inhibition assays with olive profilinˆ and polcalcin-specific rabbit polyclonal antibodies and sera from patients with chenopod allergy. cDNAs encoding these proteins were cloned by means of PCR and sequenced. Result s : Purified Che a 2 (profilin) and Che a 3 (polcalcin) exhibited prevalences of 55% and 46%, respectively, in patients (n=104) hypersensitive to chenopod pollen. Both purified allergens individually inhibited the IgE binding to the whole pollen extract and showed strong cross-reactivity with the corresponding olive pollen profilin (Ole e 2) and polcalcin (Ole e 3). Chenopod profilin consists of a 131-amino-acid chain that displays identities of approximately 75% and 82% with pollen and food profilins, respectively. Che a 3 (86 amino acids) displays similarity (65% to 82% identity) with polcalcins from pollens of olive, birch, alder, rapeseed, and timothy. Conclusion : Profilin and polcalcin are relevant panallergens in chenopod pollen and good candidates to be involved in IgE cross-reactivity with other pollen sources, thus explaining the highly frequent polysensitization of patients allergic to chenopod.

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[41] - Pecora S, Burastero S, Milani M, Asero R, Pagani M. Clinical relevance of profilin sensitisation detected by skin prick test: a multicenter prospective study in patients with grass pollinosis. Allergy 2009;64(Suppl. 90):125

Rationale: Sensitisation to Profilin, a pan-allergen, is observed in 40% of grass allergic patients. So far the are no data regarding the clinical relevance of sensitization to profilin, in term of severity of symptoms in these patients. A purified natural date palm profilin (Pho-d-2) Skin Prick Test (SPT) has been recently developped. We investigated the prevalence of profilin sensitisation and compared symptom and symptomatic drug scores during pollen season in patients with grass rhinoconjuctivitis in relation with profilin sensitisation. Patients and Methods: 50 consecutive patients with rhinoconjunctivitis and/or asthma due to grass pollen (28 men, mean age 34 years) were enrolled. SPT for grass and profilin were evaluated before pollen season 2008. Symptom score during peak pollen season (May 2008) was rated evaluating 6-item for nose and eye symptoms using a 4-point ordinal scale (0= no symptoms; 3=severe symptoms). Medication score was evaluated assessing the use of oral/topic antihistamine or corticosteroid drugs. Results: All patients were positive for grass SPT. A total of 21 subjects (44%) were positive to profilin SPT (wheal diameter >3 mm: P+). In P + patients, seasonal symptom score was significantly (P=0.01) higher in comparison with P- subjects (155 vs. 71). Medication score, mean (SD) was 39(28) in P+ patients and 13(16) in P- subjects (P=0.0025). Conclusion: Our study confirms that in grass allergic patients profilin sensitization detected by a specific SPT is detected in 40% of the patients. In these patients profilin sensitisation is also associated with an higher symptom and medication score during pollen season.

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[42] - 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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[43] - 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

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[44] - van Ree R. Clinical importance of cross-reactivity in food allergy. Curr Opin Allergy Clin Immunol 2004;4:235-240

PURPOSE OF REVIEW: Review of recent developments in the field of cross-reactivity in food allergy and the clinical relevance of these developments. RECENT FINDINGS: New foods have been added to the list of Bet v 1 and profilin-related food allergies. Clinical relevance of cross-reactions based on recognition of carbohydrate determinants and profilin is limited for the population of pollen-allergic patients as a whole. For selected food allergic patients, however, N-glycans and particularly profilin are potentially of clinical relevance. Lipid transfer proteins have further been established as clinically more severe allergens in several foods. This severity is attributed to their stability to proteolysis and processing. Storage proteins of several nuts and seeds have been identified as important allergens, but cross-reactivity between storage proteins of different foods appears to be limited. Using cross-reactivity as the basis for immunotherapy in food allergy seems promising but needs confirmation by double-blind, placebo-controlled trials. SUMMARY: The continued identification and characterization of cross-reactive allergens facilitates the study of factors determining clinical relevance of cross-reactivity and of possible efficacy of immunotherapy in food allergy.

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[45] - Ferreira F, Hawranek T, Gruber P, Wopfner N, Mari A. Allergic cross-reactivity: from gene to the clinic. Allergy 2004;59:243-267

A large number of allergenic proteins have now their complete cDNA sequences determined and in some cases also the 3D structures. It turned out that most allergens could be grouped into a small number of structural protein families, regardless of their biological source. Structural similarity among proteins from diverse sources is the molecular basis of allergic cross-reactivity. The clinical relevance of immunoglobulin E (IgE) cross-reactivity seems to be influenced by a number of factors including the immune response against the allergen, exposure and the allergen. As individuals are exposed to a variable number of allergenic sources bearing homologous molecules, the exact nature of the antigenic structure inducing the primary IgE immune response cannot be easily defined. In general, the 'cross-reactivity' term should be limited to defined clinical manifestations showing reactivity to a source without previous exposure. 'Co-recognition', including by definition 'cross-reactivity', could be used to describe the large majority of the IgE reactivity where co-exposure to a number of sources bearing homologous molecules do not allow unequivocal identification of the sensitizing molecule. The analysis of reactivity clusters in diagnosis allows the interpretation of the patient's reactivity profile as a result of the sensitization process, which often begins with exposure to a single allergenic molecule.

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[46] - 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

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[47] - Asero R, Monsalve R, Barber D. Profilin sensitization detected in the office by skin prick test: a study of prevalence and clinical relevance of profilin as a plant food allergen. Clin Exp Allergy 2008;38:1033-1037

BACKGROUND: Profilin, a pan-allergen present in all eukaryotic cells, is one of the main causes of cross-sensitization between pollen and plant-derived foods, but its clinical relevance as a food allergen is still debated . OBJECTIVE: To investigate the prevalence of profilin sensitization in a pollen-allergic population and its clinical relevance as a food allergen . METHODS: Two hundred consecutive patients with pollen allergy underwent skin prick tests (SPT) with purified natural date palm profilin (Pho d 2; 50 microg/mL; Alk Abello, Madrid, Spain). Those reporting adverse reactions to foods (confirmed by SPT with either commercial food extracts or fresh foods) underwent SPT with an apple extract containing uniquely Mal d 1 (2 microg/mL; ALK-Abello), and with a commercial peach extract containing uniquely lipid transfer protein (LTP 30 microg/mL; ALK-Abello) . RESULTS: Sixty patients (30%) showed skin reactivity to date palm profilin, Pho d 2. All were sensitized to grass pollen, and most of them reacted to birch, mugwort, ragweed and plantain pollen as well. SPT with pellitory and cypress scored negative in a high proportion of profilin reactors [26/60 (43%) and 33/60 (55%), respectively]. More than one half (34/60 [57%]) of profilin reactors had food allergy; 21 of these were monosensitized to profilin, 11 were sensitized to both profilin and Bet v 1 homologous protein, one to both profilin and LTP, and one to all the three allergens. The large majority of profilin-allergic patients reported oral allergy syndrome as the only food-induced symptom and were able to tolerate the offending foods if they were cooked or otherwise processed. Twenty-eight of 34 reported reactivity to two or more plant-derived foods. Rosaceae, tree nuts, melon and watermelon, tomato, pineapple, citrus fruits and banana were the more frequently offending foods . CONCLUSION: Profilin should be considered a clinically relevant food allergen. Allergy to melon, watermelon, tomato, banana, pineapple and orange may be considered as a marker of profilin hypersensitivity. This study underlines the clinical importance of being able to diagnose hypersensitivity to single food allergenic proteins by SPT, particularly when the relevant food allergen sources contain several allergens that show different chemical/physical features and, hence, completely different risk profiles.

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[48] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[49] - Fernandez-Rivas M, Benito C, Gonzalez-Mancebo E, Alonso Diaz de Durana MD. Allergies to fruits and vegetables. Pediatr Allergy Immunol 2008;19:675-681

Allergic reactions to fruits and vegetables are frequently observed in older children and adolescents. They can result from a primary sensitization to food allergens or from a primary sensitization to inhalant allergens such as pollens or latex. In the case of fruit allergies, the stability of the allergens involved is crucial to the sensitization pathway and in the clinical presentation of the food allergy. Two patients allergic to fruits are presented and discussed in the light of the allergens involved. Patient 1 was a 14 yr-old girl with a grass and olive pollen allergy who developed oropharyngeal symptoms typical of the oral allergy syndrome (OAS) with multiple fruits from taxonomically unrelated families, and who was sensitized to profilin. Patient 2 was an 8 yr-old girl, with no pollen allergies, who developed systemic reactions to peach and apple, and who was sensitized to non-specific lipid transfer proteins (LTP). Profilins are labile allergens present in pollens and foods, and sensitization occurs through the respiratory route to pollen profilin. The cross-reactive IgE antibodies generated can elicit local reactions in the oropharyngeal mucosa (OAS) when exposed to fruit profilins. In contrast, LTPs are a family of stable allergens that resist thermal treatment and enzymatic digestion, and can thus behave as true food allergens inducing primary (non-pollen related) sensitizations and triggering systemic reactions. These two cases represent two distinct patterns of sensitization and clinical expression of fruit allergies that are determined by the panallergens involved (LTPs and profilins) and their intrinsic physicochemical properties. Additionally, these two cases also show the improved diagnostic value of Component Resolved Diagnosis, and strengthen its utility in the routine diagnosis and management of patients.

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[50] - Fernández-Rivas M, Barreales L, Gonzalez-Mancebo E, Zuidmeer L, Mills C, Hofmann-Sommergruber K, et al. Specific IgE Responses To Lipid Transfer Protein (LTP) And Profilin Have Interactive Effects On The Outcome Of Doubleblind Placebo-controlled Food Challenges (DBPCFC) With Apple In Allergic Patients From Spain. J Allergy Clin Immunol 2008;121:S240

RATIONALE: We aimed to study the relationship of the IgE responses to the apple allergens Mal d 1-2-3-4, and the outcome of DBPCFC with apple. METHODS: In 92 Spanish patients who reported immediate reactions to apple and had positive prick-prick tests with fresh apple, the IgE responses to Mal d 1-2-3-4 were analysed by RAST, and reactivity to apple was assessed by DBPCFC. The relationship between RAST to apple allergens and outcome of DBPCFC was analysed by logistic regression. RESULTS: IgE responses to Mal d 3 (LTP) and Mal d 4 (profilin) were significantly associated with DBPCFC outcome, although an interaction was found. The reference category for risk estimation was the presence of negative (<0.30 IU/ml) RASTs to Mal d 3 and 4 that carried over a probability of a positive DBPCFC of 25%. With a negative Mal d 3 and a positive Mal d 4 the probability of a positive DBPCFC was 85.7% (risk ratio [RR] 3.43, 95% confidence interval [CI]: 1.01-11.60; p = 0.008). With a positive Mal d 3 and a negative Mal d 4 the probability of a positive DBPCFC was 90.3% (RR 3.61, 95% CI: 1.08-12.06; p = 0.001). With positive Mal d 3 and 4 the probability of a positive DBPCFC was 91.7% (RR 3.25, 95% CI: 1.13-9.39; p = 0.0003). CONCLUSIONS: Apple LTP and profilin are clinically relevant allergens in Spanish patients, and they have an interactive effect on the clinical reactivity to apple since the presence of specific IgE to them separately or in combination carries over a similar probability of positive DBPCFC.

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[51] - Rodriguez-Perez R, Crespo JF, Rodríguez J, Salcedo G. Profilin is a relevant melon allergen susceptible to pepsin digestion in patients with oral allergy syndrome. J Allergy Clin Immunol 2003;111:634-639

BACKGROUND: Melon allergy has been documented by means of double-blind, placebo-controlled food challenges. The most common clinical feature associated with melon allergy is oral allergy syndrome (OAS). However, no relevant allergens of melon have been identified to date . OBJECTIVE: We sought to identify melon allergens and analyze their digestibility in human saliva and simulated gastric fluid (SGF) to provide a rationale for the OAS . METHODS: Melon, zucchini, cucumber, and watermelon allergens were identified by means of IgE immunoblotting of sera from 21 patients with OAS after melon ingestion confirmed by means of double-blind, placebo-controlled food challenge. Further characterization was performed with rabbit antisera against sunflower pollen profilin and anticomplex glycans. Lability of allergens was assayed by incubation of melon extract in human saliva and SGF . RESULTS: Several IgE-binding components between 15 and 60 kd and a main reactive band of 13 kd were detected in melon extract with the pooled sera from patients with melon allergy. As in melon, 13-kd components of zucchini, cucumber, and watermelon extracts were strongly recognized by the IgE antibodies of the patients with melon allergy and were identified as profilins. Putative cross-reacting carbohydrate determinants were also detected. Sera from 71% of patients recognized the melon profilin, and therefore profilin is considered a major allergen. Melon allergens were unaffected by crude human saliva. In contrast, most melon proteins, predominantly the 13-kd component (profilin), were quickly digested in the SGF . CONCLUSION: In patients with OAS, a 13-kd protein identified as a profilin is a major melon allergen highly susceptible to pepsin digestion.

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[52] - Sirvent S, Castro L, Prado N, Batanero E, Torres M, Mas S et al. Digestion stability of mustard allergens: implications on their allergenicity. Allergy 2010;65(suppl. 92):713

Background: Food allergens sensitization via gastrointestinal tract requires contact to gut immune system in an immunologically active form. Thus, the resistance to digestion seems to be related with the allergenic potential of food proteins. Mustard is one of the most significant allergenic spices in terms of potency and widespread use. Sensitization to mustard is currently associated with severe symptoms. In this work, we studied the effect of gastric and duodenal in vitro digestion on the Sin a 1 (2S albumin), Sin a 3 (nsLTP) and Sin a 4 (profilin) IgE-reactivity, in order to analyzed the relationship between the severity of the symptoms associated to these allergen families and their resistance to digestion. Methods: Mustard allergens Sin a 1, Sin a 3 and Sin a 4 were purified according to established methods. Digestion was assessed individually in simulated gastric or intestinal fluid as a model of the stomach and duodenum digestion. SDS-PAGE and Coomassie staining were used to detect the digestion products. Allergenicity was analyzed by western blot using sera from patients allergic to mustard. BSA was also included as control. Results: Gastric digestion of Sin a 3 produced fragments that were visible up to 2 h. For Sin a 1 no significant fragments were detected after 2 h of incubation, although it was degraded in duodenal digestion, as occurred to Sin a 3. Sin a 4 and BSA were rapidly processed by gastric enzymes but proteins remain intact after duodenal digestion. Sin a 1 and Sin a 3 were immunologically active after 2 h of protease treatment. In contrast, Sin a 4 IgE-reactivity was abolished within less than 1 min of gastric digestion. Duodenal treatment reduces IgE-binding capability of the three allergens after 30 min. Conclusion: Sin a 1 and Sin a 3 are stable to gastric and duodenal digestion in vitro, keeping its IgE-binding capability. Therefore, both allergens can be defined as ‘true food allergens’, triggering severe reactions by reaching the gut immune system. In contrast, Sin a 4 can be considered as ‘false food allergen’ since its IgE-reactivity was abolished by gastric digestion, as was reported for other pollen-related food allergens.

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[53] - Ma Y, Zuidmeer L, Bohle B, Bolhaar STH, Gadermaier G, Gonzalez-Mancebo E, et al. Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy. Clin Exp Allergy 2006;36:1087-1096

BACKGROUND: Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear . OBJECTIVE: The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component-resolved allergy diagnosis . METHODS: Complementary DNA-derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l-proline) sepharose. A total of 28 sera from apple-allergic patients were used for IgE-ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients . RESULTS: Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small-angle X-ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion . CONCLUSIONS: Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component-resolved diagnosis of food allergy.

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[54] - Lopez-Torrejon G, Crespo JF, Sanchez-Monge R, Sanchez-Jimenez M, Alvarez J, Rodriguez J, et al. Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen. Clin Exp Allergy 2005;35:1065-1072

BACKGROUND: Melon allergy is commonly associated with oral allergy syndrome (OAS) and with hypersensitivity to pollens and other plant foods. No melon allergen responsible for these clinical characteristics has yet been isolated, although profilin has been proposed as a potential target . OBJECTIVE: To isolate natural and recombinant melon profilin, to evaluate its in vivo and in vitro reactivity, and to analyse its behaviour in simulated gastric fluid (SGF) and heat treatments . METHODS: A pool or individual sera from 23 patients, and an additional group of 10 patients, all of them with melon allergy, were analysed by in vitro and in vivo tests, respectively. Natural melon profilin (nCuc m 2) and its recombinant counterpart (rCuc m 2) were isolated by poly-l-proline affinity chromatography, and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization analysis, DNA sequencing of cDNAs encoding rCuc m 2, and immunodetection with anti-profilin antibodies. In vitro analysis included IgE immunodetection, specific IgE determination, ELISA-inhibition assays, and histamine release (HR) tests. In vivo activity of nCuc m 2 was established by skin prick testing (SPT). The effect of SGF and heat treatment on rCuc m 2 was followed by immunodetection, ELISA inhibition, and HR assays . RESULTS: Both purified forms of melon profilin were recognized by rabbit anti-profilin antibodies and IgE of sera from allergic patients, and showed molecular sizes typical of the profilin family. nCuc m 2 had a blocked N-terminus, whereas rCuc m 2 rendered the expected N-terminal amino acid sequence, its full protein sequence being highly similar (98--71% identity) to those of profilins from plant foods and pollens. The natural allergen displayed a slightly higher IgE-binding capacity than its recombinant counterpart. Specific IgE to nCuc m 2 and rCuc m 2 was found in 100% and 78% of the 23 individual sera analysed, respectively. nCuc m 2 evoked positive SPT responses in all (10/10) patients tested, and rCuc m 2 induced HR in two out of three sera assayed. SGF treatment readily inactivated rCuc m 2, as shown by its loss of recognition by anti-profilin antibodies, lack of IgE binding, and inability to induce HR. In contrast, heat treatment did not affect the IgE-binding capacity of rCuc m 2 . CONCLUSIONS: Profilin is highly prevalent in melon-allergic patients, and promptly inactivated by SGF, as expected for an allergen mainly linked to OAS.

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[55] - Scheurer S, Lauer I, Foetisch K, Moncin MS, Retzek M, Hartz C, et al. Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion. J Allergy Clin Immunol 2004;114:900-907

Background Nonspecific lipid transfer proteins (nsLTPs) have been identified as major fruit allergens in patients from the Mediterranean area. Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific biophysical and biochemical properties of this allergen family. Objective To assess the protein stability and allergenic potency of nsLTP from fruits in comparison with birch pollen-related allergens from the same allergenic source. Methods Stability of natural and recombinant cherry allergens Pru av 3 (nsLTP), Pru av 1 (Bet v 1 homologue), and Pru av 4 (profilin) to pepsin digestion and to thermal processing and stability of allergens in skin prick test reagents was investigated by immunoblotting and/or circular dichroism spectroscopy. Moreover, allergenicity of processed and fresh fruits in regard to Pru av 1 and Pru av 3 was analyzed by histamine release assays. Results Lipid transfer proteins showed the highest resistance to digestion by pepsin (rPru av 3 > rPru av 1 > rPru av 4). Immunologically active Pru av 3 was detectable after 2 hours of digestion by pepsin, whereas IgE reactivity of Pru av 1 and Pru av 4 was abolished within less than 60 minutes. In contrast with Pru av 1, IgE reactivity to nsLTPs was not diminished in thermally processed fruits, and secondary structures of purified Pru av 3 were more resistant to heating. Moreover, nsLTPs were stable components in skin prick test reagents. Histamine release assays confirmed the strong allergenicity of nsLTPs, which was not affected by protease treatment or thermal processing of fruits. Conclusion In contrast with birch pollen-related allergens, nsLTPs are highly stable to pepsin treatment and thermal processing and show higher allergenic potency. Therefore, nsLTPs have the potential to act as true food allergens, probably eliciting severe systemic reactions by reaching the intestinal mucosa in an intact and fully active form.

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[56] - Rodriguez-Perez R, Crespo JF, Rodríguez J, Salcedo G. Profilin is a relevant melon allergen susceptible to pepsin digestion in patients with oral allergy syndrome. J Allergy Clin Immunol 2003;111:634-639

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[57] - Vieths S, Reindl J, Müller U, Hoffmann A, Haustein D. Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure. Eur Food Res Technol 2000;209:379-388

Stability under digestion is thought to be an important prerequisite determining allegenicity of food proteins. To test this hypothesis, 2 allergenic plant-derived foods, protein extracts from roasted peanuts and unprocessed (native) hazelnuts, were digested by a static, 2-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. Extracts were subjected to gastric digestion for 2 h followed by a 45-min treatment under duodenal conditions. Undigested control samples and the 2 digests were investigated by SDS-PAGE, by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in mice. Peanut proteins appeared to be more stable under digestion than hazelnut proteins. The gastric digest from peanut contained various protein fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot reactivities decreased strongly after subsequent pancreatic digestion. In the gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein fragments of <15 kDa. This serum showed no binding to blots of the pancreatic digest. Sera from hazelnut-allergic patients presented IgE reactivities to an 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands >30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the 2 digests prepared from hazelnut extract. Under the native conditions of EAST, both digests from peanuts strongly reacted with human IgE. Their IgE binding capacity persisted at a level of approx. 50% when compared to undigested peanut. In the case of hazelnuts, IgE reactivity of untreated samples was reduced to <10% by both gastric and combined gastric/duodenal digestion for a serum pool prepared from 4 patients and sera from 3 additional participants. By contrast, a constantly high immunoreactivity of the hazelnut digests was detected with serum from 1 patient. EAST results were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, results indicated that the EAST and RBL cell assay are superior to immunoblotting for immunologic testing of digests. Allergenicity of peanut proteins was very persistent during digestion, whereas the native birch-pollen-related hazelnut allergens appeared to be relatively labile under identical conditions

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[58] - Jankiewicz A, Baltes W, Boegl KW, Dehne LI, Jamin A, Hoffmann A, et al. In vitro study of the gastrointestinal stability of celery allergens. Food Agric Immunol 1998;9:203-217

The gastrointestinal stability of proteins and allergens from native celery roots was studied by a simple two-step in vitro procedure. Commercial enzyme tablets that contained peptic and pancreatic enzymes were used to simulate gastric and duodenal conditions, respectively. The influence on the whole protein pattern was monitored by SDS-PAGE. The antigenicity and allergenicity of the 3 known important allergenic structures of celery were investigated with IgG from 2 rabbit antisera, IgE from sera of allergic patients with known specificity for individual allergens, and by a mediator release assay utilizing allergen-specific polyclonal murine IgE. The following allergens were studied: (i) Api g 1, a 16-kDa celery protein which shares high sequence identity with the major birch pollen allergen, Bet v 1, (ii) celery profilin that belongs to a group of ubiquitous 'plant pan allergens' and (iii) ubiquitous carbohydrate determinants present in many glycans of plant proteins in the mass range of 35-90 kDa. SDS-PAGE indicated degradation of many proteins under gastric conditions, but immunoblotting showed only a weak influence on the antigenicity and IgE reactivity of the allergens. The protein degradation was clearly enhanced after 45 min of pancreatic digestion. To a weaker degree, all allergens were again recognized on immunoblots. The presence of a considerable residual allergenic activity which seems to resist even duodenal conditions was confirmed by the determination of human IgE specific for the digests using an Enzyme Allergosorbent Test. In addition, competitive IgE inhibition assays and measuring the celery-induced mediator release of permanently growing RBL-2H3 cells that had been passively sensitized with celery-specific murine IgE showed similar results. The generation of small IgE binding peptides (<=3 kDa) was tested by ultrafiltration experiments. The whole IgE binding capacity was present in the retentates even after duodenal digestion. It is concluded that, like many other allergens from foods and other sources, the allergens of native celery are relatively stable under gastrointestinal conditions and that residual allergenic activity is due to undigested allergens.

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[59] - Jankiewicz A, Aulepp H, Baltes W, Bogl KW, Dehne LI, Zuberbier T, et al. Allergic sensitization to native and heated celery root in pollen-sensitive patients investigated by skin test and IgE binding. Int Arch Allergy Immunol 1996;111:268-278

The rates of sensitization and allergy to four birch pollen related plant foods were investigated in a group of 167 patients who were sensitive to at least one kind of pollen and one particular food. Sensitivity was concluded from a positive skin prick test or the determination of specific IgE, whereas allergy was based on anamnestic data. The positivity rates for sensitization and allergy, respectively, were: apple, 93 and 84%; hazelnut, 90 and 78%; celery, 70 and 14%; carrot, 60 and 37%. Comparative testing by skin prick test and enzyme allergosorbent test (EAST) with extract from native and microwaved (750 W, 30 min, 100 degrees C) celery root was performed on 46 of these patients. At least one positive test result (either prick test or EAST) was obtained for native celery in 36/46 (78%) and for heated celery in 20/46 (43%) of these patients. Although the concordance between the EAST and the skin test was very low, extended control experiments of both test procedures revealed no evidence for nonspecificity. Immunoblot analyses of extract from native celery and sera of 60 patients with a positive EAST (class > or = 2, > or = 0.7 U/ml) for celery resulted in the following rates of IgE binding to known cross-reactive celery allergens: Api g 1:33%, celery profilin: 17%; multiple bands most probably due to carbohydrate epitopes: 32%. The rate of binding to other allergens was below 10%. Since these three important structures are also present in birch pollen, no allergen could be identified as a candidate to mediate an exclusive celery/mugwort association. Investigation of extract from native and heated celery by immunoblotting pointed to a high lability of Api g 1, whereas profilin and carbohydrate epitopes appeared to be more resistant to heat. It has been concluded that sensitization to celery in German patients is without clinical significance in the majority of cases, in contrast to other birch-pollen-related plant foods such as apple and hazelnut. For the particular kind of extract used, neither the EAST nor the skin test alone represents an appropriate diagnostic method for testing sensitization to celery.

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[60] - 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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[61] - Lopez-Torrejon G, Crespo JF, Sanchez-Monge R, Sanchez-Jimenez M, Alvarez J, Rodriguez J, et al. Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen. Clin Exp Allergy 2005;35:1065-1072

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[62] - 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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[63] - Pali-Schöll I, Herzog R, Wallmann J, Szalai K, Brunner R, Lukschal A et al. Antacids and dietary supplements with an influence on the gastric pH increase the risk for food sensitization. Clin Exp Allergy 2010;40:1091-1098

Summary Background Elevation of the gastric pH increases the risk for sensitization against food allergens by hindering protein breakdown. This can be caused by acid-suppressing medication like sucralphate, H2-receptor blockers and proton pump inhibitors, as shown in recent murine experimental and human observational studies. Objective The aim of the present study was to assess the sensitization capacity of the dietary supplement base powder and of over-the-counter antacids. Methods Changes of the pH as well as of protein digestion due to base powder or antacids were measured in vitro. To examine the in vivo influence, BALB/c mice were fed codfish extract with one of the acid-suppressing substances. Read-out of antibody levels in the sera, of cytokine levels of stimulated splenocytes and of intradermal skin tests was performed. Results The pH of hydrochloric acid was substantially increased in vitro by base powder as well as antacids in a time- and dose-dependent manner. This elevation hindered the digestion of codfish proteins in vitro. A significant increase in codfish-specific IgE antibodies was found in the groups fed codfish combined with Rennie((R)) Antacidum or with base powder; the latter also showed significantly elevated IgG1 and IgG2a levels. The induction of an anaphylactic immune response was proven by positive results in intradermal skin tests. Conclusions Antacids and dietary supplements influencing the gastric pH increase the risk for sensitization against allergenic food proteins. As these substances are commonly used in the general population without consulting a physician, our data may have a major practical and clinical impact.

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[64] - Teuber SS. Hypothesis: the protein body effect and other aspects of food matrix effects. Ann N Y Acad Sci 2002;964:111-116

With regard to the allergenicity of edible seeds and nuts, certain proteins may not be immediately accessible to digestion in the stomach and the upper small intestine because of the nature of the organization of such proteins into protein body organelles. Protein body hydration status, interactions between proteins, phytochemicals, protease inhibitors, and other matrix effects may contribute to the ability of a protein or package of proteins to reach the sites of active immune sampling in the gastrointestinal mucosa and thus be an influence on the potential allergenicity of a protein.

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

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

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[66] - Asero R. Plant Food Allergies: A Suggested Approach to Allergen-Resolved Diagnosis in the Clinical Practice by Identifying Easily Available Sensitization Markers. Int Arch Allergy Immunol 2005;138:1-11

"BACKGROUND: Molecular biology techniques have led to the identification of a number of allergens in vegetable foods, but due to the lack of purified food proteins for routine diagnostic use, the detection of sensitizing allergens remains a nearly impossible task in most clinical settings. The allergen-resolved diagnosis of food allergy is essential because each plant-derived food may contain a number of different allergens showing different physical/chemical characteristics that strongly influence the clinical expression of allergy; moreover, many allergens may cross-react with homologue proteins present in botanically unrelated vegetable foods . OBJECTIVE: Through a review of the available literature, this study aimed to detect ""markers"" of sensitization to specific plant food allergens that are easily accessible in the clinical practice . RESULTS: There are several ""markers"" of sensitization to different allergenic proteins in vegetable foods that can be helpful in the clinical practice. Specific algorithms for patients allergic to Rosaceae and to tree nuts were built . CONCLUSION: Clinical allergologists lacking the assistance of an advanced molecular biology lab may take advantage of some specific clinical data as well as of some ""markers"" in the difficult task of correctly diagnosing patients with plant food allergy and to provide them the best preventive advice"

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[67] - Asero R, Jimeno L, Barber D. Component-resolved diagnosis of plant food allergy by SPT. Eur Ann Allergy Clin Immunol 2008;40:115-121

BACKGROUND: Fruits and vegetables may contain both labile and stable allergens. The former induce only OAS, whereas stable allergens may induce systemic reactions. Component-resolved diagnosis (CRD) of allergy to plant foods is therefore essential for the clinical management of allergic patients. METHODS: 80 adults allergic to plant foods underwent SPT with purified natural date palm profilin (Pho d 2), purified Mal d 1, a peach extract containing uniquely LTP, and with a kiwi extract containing uniquely stable allergens. RESULTS: 58 (72%) patients were monosensitized: 24 to Mal d 1, 24 to profilin, 7 to LTP, and 3 to kiwi. 22 patients were multi-sensitised: 14 to Mal d 1 and profilin, 2 to Mal d 1 and kiwi, 1 to LTP and profilin, 3 to LTP and Mal d 1, and 2 to LTP, Mal d 1 and profilin. Mal d 1 and LTP sensitisation were associated with apple and peach allergy, respectively, whereas profilin sensitisation was associated with allergy to melon, watermelon, banana, tomato and citrus fruits. 18/21 kiwi-allergic patients were sensitised to one of the cross-reacting allergens, but 2/18 reacted to kiwi-specific allergens as well. CONCLUSIONS: In patients with allergy to plant-derived foods CRD can be performed by SPT with purified allergen proteins. In the future, the availability of a larger number of purified natural or recombinant allergens for SPT will represent a simple means to classify food-allergic patients properly on the first visit.

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[68] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[69] - Rodriguez-Perez R, Crespo JF, Rodríguez J, Salcedo G. Profilin is a relevant melon allergen susceptible to pepsin digestion in patients with oral allergy syndrome. J Allergy Clin Immunol 2003;111:634-639

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[70] - Lopez-Torrejon G, Crespo JF, Sanchez-Monge R, Sanchez-Jimenez M, Alvarez J, Rodriguez J, et al. Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen. Clin Exp Allergy 2005;35:1065-1072

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[71] - Rodriguez-Perez R, Fernández-Rivas M, González-Mancebo E, Sánchez-Monge R, Diaz-Perales A, Salcedo G. Peach profilin: cloning, heterologous expression and cross-reactivity with Bet v 2. Allergy 2003;58:635-640

BACKGROUND: Peach is among the main foods causing allergic reactions in the Mediterranean adult population. Only a single peach allergen, named Pru p 3, has been characterized. However, a potential role of profilin has also been suggested in grass pollen-associated allergy to peach . METHODS: Complementary DNA clones for two different peach profilin isoforms were obtained by reverse transcriptase polymerase chain reaction using non-degenerated primers. Expression of recombinant peach profilin was performed in Escherichia coli, and confirmed using rabbit polyclonal antibodies to sunflower pollen profilin. Twenty-nine individual sera from patients with peach allergy proved by double-blind, placebo-controlled food challenges (DBPCFC), either with (n = 15) or without (n = 14) specific IgE to Bet v 2, were used in immunodetection assays to test recombinant peach profilin reactivity . RESULTS: Each peach profilin cDNA included an open reading frame coding for a 131 amino acid protein. The peach profilin isoforms, designated Pru p 4.01 and Pru p 4.02, showed 80% of amino acid sequence identity, and were very similar (>70% identity) to allergenic profilins from plant foods and pollens. Recombinant Pru p 4.01 was expressed in E. coli as a nonfusion protein, displaying the expected molecular size and reacting with anti-profilin antibodies. rPru p 4.01 was recognized by all sera (15 of 15) with specific IgE to Bet v 2, whereas no sera (zero of 14) without IgE to this birch allergen reacted with rPru p 4.01 . CONCLUSIONS: Peach profilin Pru p 4 is very closed to other allergenic profilins from plant foods and pollens. A complete correlation between reactivity to rPru p 4 and rBet v 2 has been found in sera from peach allergic patients.

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[72] - Fäh J, Wüthrich B, Vieths S. Anaphylactic reaction to lychee fruit: evidence for sensitization to profilin. Clin Exp Allergy 1995;25(10):1018-1023

Due to the increasing popularity of exotic fruits in the Western diet, allergologists are confronted with allergic reactions to substances in these plants. The present report describes an anaphylactic reaction after the consumption of lychee fruit (Litchi sinensis). The atopic patient also suffers from rhinoconjunctivitis due to a sensitization against pollen of the Compositae family, as well as from dyspnoea after eating sunflower seeds. Our goals were to determine crossreactivity between antibodies against lychee fruit and other plants and to characterize the allergen. METHODS AND RESULTS: Specific IgE against lychee fruits were detected by an EAST assay. The allergen was characterized by immunoblot, immunoblot inhibition and EAST inhibition assays. Broad crossreactivity between lychee fruit and other plants was found and profilin identified as the protein responsible for the patient's complex allergy syndrome. CONCLUSION: Lychee fruit contains a significant amount of profilin. Consumption of this exotic fruit can cause severe anaphylactic reactions in patients being sensitized against the plant pan-allergen profilin.

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[73] - Reindl J, Rihs HP, Scheurer S, Wangorsch A, Haustein D, Vieths S. IgE Reactivity to Profilin in Pollen-Sensitized Subjects with Adverse Reactions to Banana and Pineapple. Int Arch Allergy Immunol 2002;128:105-114

Background: The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies. By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization. Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits. Methods: Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers. The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells. The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase. IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST). The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments. Results: Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%). IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments. Conclusions: Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits

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[74] - Nilsson N, Dahlström J, Lilja G, Nilsson H, Lidholm J, Borres M, et al. Grass and wheat cross reactivity - What does the component pattern look like ? Allergy 2008;63(suppl. 88):166-167

Background: IgE sensitisation to wheat is common among patients with food related symptoms but is not always associated with allergic symptoms. The sensitisation route may be both oral and by inhalation, and the clinical reactions to wheat can be triggered both by inhalation and ingestion. For example, wheat belongs to the grass family Triticae and patients with IgE antibodies to grass pollen often display antibodies towards different cereals. It is often not possible to tell, without challenge, if the patient has a food allergy to wheat or if the IgE ab reactivity reflects a sensitisation to wheat or a cross-reaction to pollen allergens. Material and methods: Ninety children (22 female and 68 male) at the age of 5 year and 10 month to 16 year and 9 month with a confirmed grass-pollen allergy were recruited consecutively. The presence of allergic disease was evaluated and serum samples were analysed with ImmunoCAPTM for allergen specific IgE antibodies to timothy grass and allergen components Phl p 1, 2, 4, 5, 6, 7, 11, 12 from timothy, bromelin (CCD), wheat and omega-5 gliadin from wheat. The data was evaluated using a treebased regression model. Results: All 90 children were IgE sensitised to timothy grass and 42/90 were also sensitised to wheat. The component which best explained the level of IgE to wheat was bromelin. Among children with low (or no) level of IgE to bromelin, IgE level to wheat was best explained by the level of IgE to Phl p. 12 (profilin). Conclusion: sensitisation to bromelin was only present in children with allergen specific IgE to wheat. If bromelin and profilin could be clinically useful to discriminate patients with a food allergy to wheat from a sensitisation without symptoms needs to be further investigated.

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[75] - Kazemi-Shirazi L, Niederberger V, Linhart B, Lidholm J, Kraft D, Valenta R. Recombinant Marker Allergens: Diagnostic Gatekeepers for the Treatment of Allergy. Int Arch Allergy Immunol 2002;127:259-268

During the past decade an increasing number of recombinant allergens have become available, representing a significant proportion of the epitope complexity of natural allergen extracts. Component-resolved diagnosis with recombinant allergens reveals the antibody reactivity profile of allergic patients and identifies the disease-eliciting allergen molecules. This article exemplifies how recombinant allergen molecules with high cross-reactive potential can be used as marker allergens to identify allergic patients who are cross-sensitized to a variety of allergen sources. It further demonstrates how the use of allergens with a restricted distribution in a certain group of allergen sources may allow the identification of patients who have been genuinely sensitized by a particular allergen molecule. Drawing from those examples, it is suggested how diagnostic tests based on such recombinant marker allergens may be used to improve the choice and monitoring of currently available forms of specific immunotherapy.

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[76] - 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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[77] - 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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[78] - 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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[79] - Valenta R, Twaroch T, Swoboda I. Component-resolved diagnosis to optimize allergen-specific immunotherapy in the Mediterranean area. J Investig Allergol Clin Immunol 2007;17(suppl. 1):88-92

Allergen-specific immunotherapy (SIT) is the only allergen-specific treatment for allergy. It can prevent progression of the disease and has a long-lasting therapeutic effect. Since SIT is allergen-specific, the identification of the disease-eliciting allergen is an essential prerequisite for the accurate prescription of treatment. Diagnostic tests based on allergen extracts consist of mixtures of various allergens of which some are specific for the allergen source and others occur as cross-reactive allergens in various unrelated allergen sources. It may therefore be difficult and sometimes impossible to identify the disease-causing allergen with such tests, particularly in patients who are sensitized to more than one allergen source. Sensitization to pollens from olive, grasses, and Parietaria in the Mediterranean area is frequently treated with SIT. Here, we describe allergen molecules from these sources that can be used for component-resolved diagnosis of allergy to facilitate the selection of patients for SIT and monitor the immunological effects of treatment.

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[80] - Focke M, Marth K, Flicker S, Valenta R. Heterogeneity of commercial timothy grass pollen extracts. Clin Exp Allergy 2008;38:1400-1408

BACKGROUND: The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources . OBJECTIVE: To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity . METHODS: Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients . RESULTS: The allergen extracts showed broad variations in protein compositions and amounts (24.1-197.7 microg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32-384 ng/mL; Phl p 2: 1128-6530 ng/mL, Phl p 5: 40-793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients . CONCLUSIONS: Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.

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[81] - 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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[82] - Asero R, Jimeno L, Barber D. Preliminary Results of a Skin Prick Test–Based Study of the Prevalence and Clinical Impact of Hypersensitivity to Pollen Panallergens (Polcalcin and Profilin. J Investig Allergol Clin Immunol 2010;20:35-38

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[83] - Barber D, de la Torre F, Lombardero M, Antépara I, Colas C, Dávila I et al. Component-resolved diagnosis of pollen allergy based on skin testing with profilin, polcalcin and lipid transfer protein pan-allergens. Clin Exp Allergy 2009;39:1764-1771

Background Allergy diagnosis needs to be improved in patients suffering from pollen polysensitization due to the existence of possible confounding factors in this type of patients. Objective To evaluate new diagnostic strategies by comparing skin responses to pan-allergens and conventional allergenic extracts with specific IgE (sIgE) to purified allergen molecules. Methods One thousand three hundred and twenty-nine pollen-allergic patients were diagnosed by a combination of an in vitro method with a panel of 13 purified allergens, including major allergens and pan-allergens, using a high-capacity screening technology (ADVIA-Centaurs) and skin prick test (SPT) to pan-allergens and conventional extracts. Results There was a high concordance (k index) between in vitro (sIgE to major allergens) and in vivo (SPT to conventional extracts) methods in patients who were not sensitized to panallergens, but SPT with conventional extracts failed to diagnose patients with sensitization to pan-allergens. In patients who were simultaneously sensitized to polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. There was a statistical association between sensitization to profilins and/or lipid transfer proteins and food allergy (Po0.0001). Conclusion The novel diagnostic strategy has proven to be a valuable tool in daily clinical practice. Introduction of routine SPT to pan-allergens is a simple and feasible way of improving diagnostic efficacy. Patients sensitized to pan-allergens should be tested by an adequate panel of allergenic molecules in order to identify the allergens that are responsible for the allergic disease.

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[84] - Kazemi-Shirazi L, Niederberger V, Linhart B, Lidholm J, Kraft D, Valenta R. Recombinant Marker Allergens: Diagnostic Gatekeepers for the Treatment of Allergy. Int Arch Allergy Immunol 2002;127:259-268

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