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Les fruits à coque : généralités

dimanche 4 mai 2008, par Allerdata

Qu’est-ce qu’un « fruit à coque » ?

A priori, cette notion renvoie à la présence d’une enveloppe scléreuse entourant la graine. C’est la cas des graines issues de drupes, c’est-à-dire de fruits charnus à noyau (ex. amande, noix, etc..). On devrait donc y inclure des graines qui ne sont pas rangées habituellement dans les fruits à coque, comme la noix de muscade ou la fève tonka (Dipteryx odorata).

Mais l’enveloppe entourant la graine est parfois semi-rigide (ex. châtaigne et faîne qui sont botaniquement des akènes) ou nue (ex. noisette, gland de chêne qui sont des mucules).

Ou la chair du fruit est séparée de la graine (ex. noix de cajou)…de sorte que les fruits à coque ne peuvent être assimilés aux graines provenant de fruits charnus renfermant un noyau rigide.

Ce ne sont pas mieux des « fruits secs », car il faudrait y inclure les raisins secs ; ni même des fruits à proprement parler !…

La dénomination de « fruits à coque » rassemble donc artificiellement des graines d’origine et d’usages très différents :

  • noisette, noix, amande, noix de cajou, etc.. proviennent de familles botaniques variées
  • si certaines de ces graines sont consommées telles quelles (ex. à l’apéritif), d’autres graines sont utilisées dans des préparations alimentaires où elles peuvent se trouver « cachées » (noisette, amande, etc..)
  • le traitement thermique qu’ont subi ces graines peut varier lui aussi.

Les anglophones rangent sous le terme de « nuts » un ensemble de graines comprenant les fruits à coque mais aussi l’arachide, de sorte que certaines statistiques sont d’interprétation difficile, les fruits à coque (FaC) n’étant pas distingués d’une cause majeure d’allergie à elle seule, l’arachide.

La terminologie anglo-saxonne renvoie même parfois à un produit qui n’est pas une graine, comme le « tiger nut » qui est en fait le rhizome tubéreux du souchet (Cyperus esculentus) .

L’expression « tree nuts » est plus restrictive : les graines provenant d’espèces à végétation herbacée, comme l’arachide, le sésame ou le tournesol, n’en font pas partie . Cependant sont parfois placés dans cette catégorie des graines qui ne sont pas des FaC, tel le pignon de pin .

Le cas de la châtaigne, dont l’intérêt allergologique la rapproche plus du latex que des FaC, est abordé ailleurs (cf. Châtaigne).


Le tableau ci-après précise l’origine botanique des principaux FaC. Il n’est pas exhaustif car de nombreuses autres graines peuvent être consommées localement ou exceptionnellement : noix de nangaille, noix d’hickory, amande de l’abricot , amande de la mangue , gland de chêne , etc..

{{}} Organisme source Famille botanique
Amande Prunus dulcis Rosacées
Noisette Corylus avellana Bétulacées
Noix Juglans regia, etc.. Juglandacées
Noix de pécan Carya illinoinensis Juglandacées
Noix de cajou Anacardium occidentale Anacardiacées
Pistache Pistacia vera Anacardiacées
Noix de macadamia Macadamia integrifolia Protéacées
Noix du Brésil Bertholletia excelsa Burséracées
Noix de coco Cocos nucifera Arecacées

A cette diversité botanique s’ajoute la variété des modes possibles de sensibilisation aux fruits à coque : multiplicité des allergènes + paramètres géographiques. Aussi, au sein des chapitres de l’allergologie moléculaire, celui concernant les fruits à coque est l’un des plus riches sur le plan des interactions moléculaires et de l’épidémiologie comparée.

De par son ampleur, ce chapitre est divisé en plusieurs thèmes et monographies, comme le détaille la liste ci-après :

Généralités

Monographies

Réactivités / allergies croisées

Allergénicité des fruits à coque

Les FaC présentent une allergénicité importante. Celle-ci est liée notamment aux différentes protéines de stockage qu’elles contiennent .

Il n’est pas exclu, par ailleurs, que cette allergénicité soit favorisée par les composants oléagineux plus ou moins constamment présents dans ces graines (cf. Allergies aux graines).

Selon les données du CICBAA, les FaC représentent 5% des allergies alimentaires chez l’enfant et 9,2% chez l’adulte. Comparativement, l’arachide est en cause chez 26% des enfants et 5,6% des adultes.

L’évolution inverse de la prévalence avec l’âge provient, en partie, de l’apparition chez l’adulte, d’une forte proportion d’allergies liées à un pollinose : la part des allergies initiées dans l’enfance s’en trouve corrélativement réduite.

De plus, certaines allergies pour des FaC sont favorisées par une pollinose, par exemple la noisette avec le bouleau.

Cette répartition est visible dans les statistiques du Réseau d’Allergo-Vigilance : sur 900 déclarations en mai 2010, 115 faisaient état de FaC, soit aussi souvent que l’arachide (111 cas) . La noix de cajou prenait la 1ère place (47 cas), devant la noisette (27 cas), la noix (13), la noix du Brésil (11), la pistache (9), l’amande (5), la noix de pécan (1), la noix de macadamia (1) et la noix de coco (1).

Une consommation limitée peut expliquer aussi la rareté des cas d’allergie pour certains FaC (ex. noix de macadamia).

En Grande-Bretagne, Pumphrey avait relevé les répartitions suivantes pour les « nuts »  :

  • anaphylaxie : arachide 41%, noix du Brésil 4%, amande 4%, noix de cajou 4%, noix 3%, noisette 3%, pistache 2%
  • décès : arachide 29%, noix 15%, noix du Brésil 9%, amande 6%, noisette 3%, mélange ou ? ? 35%

Un autre travail se rapportant à l’incidence des réactions allergiques sévères au Royaume-Uni donne des chiffres un peu différents : sur 58 cas pédiatriques, 10 étaient dus à l’arachide, 7 à la noix de cajou, 2 aux noix, 2 à la noix du Brésil et 1 à la noix de macadamia .

Et Bock, aux USA, avait relevé 10 cas dus à des FaC parmi 32 décès par allergie alimentaire .

Bien sûr, la comparaison des prévalences ou fréquences ne permet pas d’évaluer l’allergénicité intrinsèque des différents FaC. En effet, de multiples facteurs interviennent dans ces statistiques :

  • l’âge des patients
  • les pathologies intercurrentes et/ou associées : pollinose, eczéma atopique. Concernant ce dernier, il faut souligner les prévalences très élevées rencontrées dans certaines séries de patients avec allergie aux FaC, tant chez l’enfant que chez l’adulte (60 à 90%)
  • l’origine des patients peut exercer une influence à travers l’environnement pollinique mais aussi du fait d’habitudes locales de consommation des FaC :
    • aux USA, la noix arrive en tête des causes d’allergie aux FaC, devant les noix de cajou, du Brésil ou de pécan
    • en Grande-Bretagne, la noix du Brésil est la 1ère cause d’allergie aux FaC selon Clark et Ewan . La fréquence est 3 fois moindre pour la noisette, 4 à 5 fois moindre pour la noix, l’amande et la noix de cajou. Ici, une allergie aux noix de pécan est plutôt rare, contrairement aux USA
    • en Italie, Asero relève que la pistache et la noix de cajou sont peu consommées
    • une étude récente a montré que les patients Américains et Italiens allergiques à la noix avaient une réactivité à des allergènes très différents

L’histoire naturelle de l’allergie aux FaC est variable, la sensibilisation pouvant débuter très tôt dans l’enfance (ex. noix de cajou), ou du fait de l’acquisition d’une pollinose (ex. noisette), ou faisant partie d’un « syndrome LTP ».

Des facteurs propres aux patients et à leur environnement gouvernent la transition d’une réactivité infra-clinique en une allergie : ainsi Rodriguez a observé un seul cas de positivation du TPODA chez 12 patients Espagnols, réactifs en TC et/ou en CAP, au cours d’un suivi de 3-5 ans .

Inversement, Fleischer rapporte 9 TPO négatifs en suivi de 20 patients Américains qui avaient une histoire clinique positive au départ . Ceci étant, dans cette dernière étude, ainsi que dans beaucoup d’autres, la preuve de l’allergie par la réalisation d’un TPO n’avait pas été apportée au moment de l’inclusion des patients, ce qui affaiblit la conclusion des auteurs.


Fruits à coque et législation

Le décret n° 2005-944 rendant obligatoire l’étiquetage des aliments a listé les FaC concernés. On pourra remarquer l’absence de la noix de coco. Et, dans la mesure où le nom des espèces végétales est précisé dans le texte du décret, des FaC très proches mais provenant d’autres espèces pourraient sortir du cadre de l’obligation d’étiquetage.

C’est le cas pour la noix dont l’obligation ne s’applique qu’aux graines issues de Juglans regia.

Un flou existe aussi pour les noix de macadamia (auxquelles le décret ajoute les noix du Queensland comme synonyme), ces noix étant associées à un nom d’espèce végétale non accepté, Macadamia ternifolia, alors que le nom valide est M. integrifolia.

Le cas de la noix de nangaille est différent, celle-ci s’étant vue refusée l’autorisation de mise sur le marché en Europe.

Outre-Atlantique, le Food Allergy & Anaphylaxis Network (FAAN) publie des alertes de présence inopinée de composants dans les aliments manufacturés. Les FaC y tiennent une bonne place (cf. les statistiques FAAN)

Effets de la chaleur et de la digestion sur l’allergénicité des fruits à coque

Tout d’abord, quelques points doivent être soulignés :

  • les travaux de stabilité ont principalement concerné la noisette. Des FaC importants comme la noix ou la noix de cajou n’ont pas été étudiés, à notre connaissance.
  • les résultats des travaux de stabilité ont souvent une signification clinique mal établie :
    • les tests ont été effectués sur de protéines pures, isolées non seulement de leur milieu d’origine mais sans tenir compte non plus, pour la digestibilité, des conditions réalistes. Par exemple : effet matrice , autres composants du bol alimentaire , cuisson avant digestion , pH gastrique réel , etc..
    • le fractionnement d’une protéine par les enzymes digestives ne signifie pas la perte de tout pouvoir réactogène ni immunogène . Par exemple, la vérification de la disparition de l’IgE-réactivité n’est pas suffisante avec les techniques en blot car des peptides de 3-6 kD ne sont pas vus avec cette technique
    • il a été montré qu’il existait autant d’allergènes instables que d’allergènes résistant à une épreuve de digestion gastrique simulée
    • les preuves d’une modification de l’allergénicité ont rarement été recherchées par des tests in vivo
  • enfin, dans le cas d’aliments contenant plusieurs catégories d’allergènes, comme c’est le cas pour les FaC, il faudrait tenir compte des réactivités initiales au niveau moléculaire pour interpréter correctement le résultat global sur l’aliment. Par exemple, le résultat global peut varier entre patients de pays différents.


Amande

Des résultats très fragmentaires sont disponibles.

Divers procédés de chauffage (grillage, autoclavage, micro-ondes) entraînaient une diminution de l’extractabilité des protéines mais pas une perte de stabilité .

Tawde a montré une baisse de l’IgE-réactivité de l’amandine après chauffage , tandis que Bargman observait cet effet sur certaines fractions de l’amande (15 et 70 kD) mais pas sur d’autres (45-50 kD) .

De même une étude ne montrait pas de modification significative d’IgE-réactivité après chauffage .

En digestion pepsique artificielle l’amande montre une certaine résistance . Ces résultats obtenus en SDS-PAGE ne préjugent pas de l’effet de la digestion sur l’IgE-réactvité.

Cependant, l’apparition d’une néo-IgE-réactivité pour l’amande a été montrée dans cette étude pour quelques patients placés sous médication anti-ulcéreuse, ce qui montre qu’au moins pour ces patients la digestibilité de l’amande est incomplète.


Noisette

Chaleur

Deux travaux concordent pour conclure que le grillage des noisettes (par ex. 140°C 40 min) entraîne une disparition de l’IgE-réactivité de la PR-10 Cor a 1.04, tandis que des protéines de 30 kD et plus résistent bien .

Mais Wigotzki remarque qu’une bande <14 kD conserve une IgE-réactivité après 15 min à 185°C et qu’un chauffage de 90 min à 100°C seulement ne modifie pas l’IgE-réactivité globale de la noisette .

Chez des sujets Italiens positifs pour une bande 9 kD (LTP) la chaleur ne diminuait pas l’IgE-réactivité . De même, chez des patients Autrichiens non polliniques, le grillage des noisettes n’affectait pas l’IgE-réactivité < 10 kD .

Aux Pays-Bas, Wensing observe la négativation de TPODA (n=4 patients , mais Akkerdaas constate qu’une IgE-réactivité subsiste pour la 2S albumine , si bien que 12% des sujets ont encore des tests in vitro positifs pour la noisette après chauffage + digestion artificielle .

Skamstrup-Hansen note le maintien de 5 TPODA positifs après grillage des noisettes parmi 17 patients Danois et Suisses . Il est intéressant de noter que ces patients étaient avant tout réactifs pour la PR-10, Cor a 1.04, peu pour la profiline Cor a 2 (7/17) et pas du tout pour la LTP Cor a 8.

On peut supposer que Cor a 1.04 était donc en partie protégé de l’effet de la chaleur par la matrice environnante dans la noisette au cours du chauffage.

Au total, le grillage diminue l’allergénicité des noisettes chez des patients avec allergie croisée bouleau-noisette, mais pas chez tous, et n’affecte que peu l’allergénicité des noisettes si la sensibilisation est différente (LTP et/ou protéines de stockage).

Digestion

L’origine des patients, c’est-à-dire leur mode de sensibilisation à la noisette, influe sur les résultats de la digestion :

  • perte d’IgE-réactivité après digestion artificielle (gastrique + pancréatique) de noisettes fraîches chez des patients Allemands . mais une bande 10-12 kD résiste chez un patient
  • cette perte d’IgE-réactivité chez des patients par ailleurs polliniques au bouleau est attribuée à la fragilité de la PR-10 Cor a 1.04 : en digestion gastrique simulée l’IgE-réactivité de Cor a 1.04 disparaît en 1 seconde
  • dans le même travail, il a été montré que Cor a 1.04 résistait très bien, par contre, à la digestion trypsique. Aussi, les résultats obtenus par Untersmayr revêtent une grande importance : en cas d’hypo- ou d’achlorhydrie la digestion gastrique peut être incomplète et des allergènes non dégradés traverser la barrière intestinale .

Un suivi de 153 patients sous médication anti-ulcéreuse pendant 3 mois a révélé une néo-réactivité pour la noisette chez 5 d’entre eux, dont 3 TPO positifs et 2 cliniquement allergiques .

  • De fait, si le pH était porté à 5 au lieu de 2 ou 1,2 comme dans les tests classiques de digestibilité, l’IgE-réactivité n’était pas modifiée après 2h de contact avec la pepsine .

Enfin, Akkerdaas a étudié la digestion pepsique d’un extrait de noisette  :

  • si des patients Espagnols conservaient, sans surprise, leur IgE-réactivité après digestion, c’était le cas aussi pour 2 des 13 patients Milanais et pour 22 des 72 Néerlandais.
  • Les auteurs d’en conclure : il ne faut pas résumer la réactivité de la noisette à la seule PR-10, même en zone propice à une pollinose au bouleau.


Noix

On possède très peu de connaissances au sujet de la noix.

Un travail a montré une grande résistance à la digestion pepsique d’un extrait de noix, sur la base de résultats en SDS-PAGE (pas en blot avec des sérums de patients) .


Noix de coco

Fernandez-Rivas a montré que la 7S globuline ne résistait pas à 1 minute de digestion gastrique .


Noix de macadamia

Le grillage ne modifie pas la réactivité cutanée de la noix de macadamia .


Noix de pécan

Berrens et Malanin ont décrit la possibilité d’une formation de néo-allergènes au cours du grillage des noix de pécan.

La formation de composés de Maillard et/ou une modification de la digestibilité des protéines pourrait résulter du grillage des noix de pécan.

Un phénomène similaire aurait lieu en cas de conservation prolongée des noix de pécan à 30°C.

La nature de ces néo-allergènes n’a pas été élucidée, mais des observations ont montré que certains patients réagissaient seulement si les noix de pécan étaient grillées .

La stabilité a aussi été étudiée par l’équipe de Teuber, mais à l’aide d’anticorps de lapin .


Noix du Brésil

Les travaux ont porté sur le principal allergène, Ber e 1, plutôt que sur la graine elle-même.

Ils concordent tous et attribuent à cette 2S albumine une très forte stabilité à la chaleur et à la digestion .

La compacité de la protéine, ses ponts disulfures internes, des points de protéolyse peu accessibles, un très bon refolding lors du refroidissement après chauffage, la capacité de former des émulsions et l’insensibilité aux surfactants, autant d’explications qui ont été avancées pour expliquer cette stabilité .

Diagnostic d’une allergie aux fruits à coque

La pratique d’un TPODA est rare dans le cas des FaC (ex. ).

Souvent, un TPO et/ou un TPL n’est pratiqué que pour un fraction des patients .

Ewan estime que le TPO n’est pas nécessaire

  • si l’histoire clinique est convaincante et qu’elle est associée à un TC positif
  • ou si les symptômes ne sont pas aigus
  • ou si le FaC est toléré malgré un TC positif.

Inversement, le TPO est présenté comme utile pour tester une acquisition de tolérance, si le TC s’est négativé dans le suivi .

Fleischer montre que les ¾ des TPO réalisés sur la base d’une simple positivité en TC et/ou en CAP s’avèrent négatifs .

Le nombre de TC positifs pour les FaC dépasse de beaucoup celui des allergies vraies correspondantes . Par exemple, les données du CICBAA révèlent 18% de TC positifs parmi les 523 qui ont été pratiqués avec la noix du Brésil, mais seulement 5 confirmations de cette réactivité cutanée en TPO (J-M. Renaudin, comm. pers., 07/03/09). Même si des TPO n’ont pas été proposés à tous les patients TC positifs du fait de la prise en compte de l’histoire clinique également, il est clair que la réactivité cutanée pour ce FaC ne se traduit que partiellement en une allergie vraie.

Les tests in vitro pour la noix de cajou ne renseignent pas sur la gravité potentielle d’une réaction clinique . Dans l’étude de Giovanni-Chami, ni le CAP, ni le TC ne différenciaient les sujets allergiques des sujets asymptomatiques .

Une interférence due à des CCD est à craindre car dans la même étude 36% des enfants négatifs en TC étaient positifs en CAP.

Plusieurs paramètres influençant les tests diagnostiques ont été étudiés dans le cas de la noisette :

  • ni le résultat d’un TC natif, ni celui d’un CAP, ne prédisaient fiablement la réponse lors d’un TPODA chez des patients ayant une réactivité à la noisette associée à une sensibilisation au bouleau
  • les extraits commerciaux de noisette présentaient des contenus très variables en PR-10 et profiline, et la réactivité cutanée était plutôt en rapport avec le contenu total en protéines, quelles qu’elles soient, dans l’extrait
  • on peut diminuer la perte de PR-10 lors de l’obtention des extraits de noisette, mais les procédés chimiques employés sont peu compatibles avec une utilisation in vivo
  • cette fragilité de la PR-10 de la noisette, Cor a 1.04, a conduit à renforcer le contenu des extraits pour le CAP par adjonction du recombinant rCor a 1.04. Ceci a amélioré la sensibilité diagnostique du test dans certains pays … mais pas en Espagne !
  • le contenu limité en Cor a 1.04 pourrait expliquer des TC négatifs pour la noisette mais positifs, chez les mêmes sujets, pour le bouleau et le noisetier
  • certaines protéines peuvent être difficiles à extraire, ou bien être perdues au moment de la phase initiale de délipidation de la graine (ex. les oléosines )
  • un extrait effectué à pH 2,5 récupère mieux la LTP, Cor a 8, mais pas la PR-10
  • le contenu en lipides de l’aliment-test lors du TPO semble jouer un rôle sur le délai d’apparition des symptômes . Ceci étant, van der zee a montré que la dose déclenchante lors d’un TPO à la noisette variait dans un facteur de 3 au maximum à 6 mois/ 1 an de distance, chez le même sujet

On le voit, beaucoup de paramètres sont en jeu et, quand la pratique d’un TPO n’est pas réalisable, d’autres critères diagnostiques sont nécessaires.

Des seuils discriminants ont été proposés par Clark et Ewan  : un diamètre de 8 mm ou plus, en test cutané, est suffisant pour assurer le diagnostic d’allergie (9 mm pour la noix du Brésil).

Une étude plus récente de l’équipe de Hill confirme en partie cette approche et a le mérite d’avoir pris comme référence un TPO positif .

  • Selon ces auteurs, 56% des TPO pourraient être ainsi évités devant un TC égal ou supérieur à un seuil donné : les meilleurs résultats sont obtenus pour la noix du Brésil (8 mm) et la noix de pécan (7 mm), les likehood ratios étant respectivement de 15 et de 12,5. avec au moins 8 mm pour la noix, la noisette ou la noix de cajou une VPP de 100% est atteinte,
  • mais la VPN n’est pas optimale (54%, 79%, 42%). la pistache (6 mm, en natif) et surtout l’amande (7 mm) donnent des likehood ratios insuffisants (5,4 et 4,2).

Clark et Ewan ont aussi proposé un seuil de 15 kU/l en CAP comme suffisant pour poser un diagnostic d’allergie aux « nuts » en général, c’est-à-dire aussi bien pour les FaC que pour l’arachide . Cependant leurs résultats révèlent :

  • 76% des cas où un diagnostic d’allergie a été retenu sont dans une zone grise (< 15 kU/l)
  • 70% des cas présentant des symptômes sévères ont un CAP < 15 kU/l (et même 22% ont un CAP négatif)
  • inversement, 40% de CAP sont trouvés positifs pour d’autres « nuts » chez des sujets allergiques à une seule « nut »

Cette étude, bien que basée sur un nombre élevé de patients, montre en fait l’intérêt de recourir à des tests plus discriminants, comme ceux utilisant des allergènes recombinants. Ces derniers ont notamment l’avantage de pouvoir éviter l’interférence des CCD (cf. plus loin).

Pour le diagnostic d’une réactivité à la noix du Brésil, les TC ont été montrés plus pertinents que le CAP, un résultat égal ou supérieur à 6 mm avec un extrait commercial ayant une VPP de 100% .

L’introduction d’un test in vitro pour le recombinant rBer e 1 est susceptible cependant d’améliorer les performances du CAP.

A cela deux raisons : l’absence d’interférence par les IgE anti-CCD et la bonne spécificité de Ber e 1. En effet, les réactivités croisées entre Ber e 1 et d’autres 2S albumines sont limitées par une faible homologie séquentielle (ex. 28% avec Bra n 1 du colza ).

Asero a proposé un arbre décisionnel pour aider au diagnostic d’une allergie aux FaC et aux Rosacées . La démarche comprend, dans l’ordre :

  • des TC noix et noisette natifs,
  • puis un TC commercial pour la pêche (réactivité LTP),
  • puis des TC noisette et noix avec des extraits commerciaux,
  • un TC bouleau et
  • éventuellement des tests rBet v 1 et rBet v 2.

Cette approche explore donc plusieurs modes de réactivité : les LTP, les PR-10 (bouleau et rBet v 1) et les protéines de stockage des graines (TC commerciaux).

Dans la mesure où un test in vitro pour la LTP de noisette est à présent disponible (rCor a 8), l’algorithme pourrait comprendre des TC pour les FaC et une mesure de réactivité à rBet v 1 et à rCor a 8.

La possibilité de tester rCor a 8, et non plus seulement rPru p 3 comme avant, permet de solutionner une difficulté soulevée par des résultats obtenus aux Pays-Bas :

  • Dans un premier travail, il avait été remarqué que des enfants pouvaient présenter un TPODA noisette positif sans réactivité pour le bouleau, ce qui est plutôt inattendu dans cette région de l’Europe .
  • Par la suite, les mêmes auteurs ont observé que la réactivité clinique de ces enfants était très dépendante d’une positivité pour la LTP de la noisette, Cor a 8 : à l’aide d’un allergène purifié, nCor a 8, ils n’ont trouvé aucun résultat positif chez les 12 enfants négatifs en TPODA ; en cas de syndrome oral au cours du TPODA, 1 enfant sur 4 était positif pour nCor a 8 ; et si des symptômes objectifs se faisaient jour au cours du TPODA, c’était cette fois tous les enfants qui étaient Cor a 8 positifs .

Le problème posé par cette étude tenait dans le peu de représentativité de la LTP de pêche chez ces enfants Néerlandais : rPru p 3 n’était positif que pour 2 des 9 enfants positifs pour nCor a 8.

  • Pour ces derniers, la LTP de noisette, c’est-à-dire la LTP de l’aliment suspecté de provoquer les réactions allergiques, était plus pertinente que rPru p 3, la LTP « modèle ».

Un autre travail, bien que purement sérologique, avait suggéré cette limitation :

  • à partir d’une banque de sérums, il avait été montré une meilleure corrélation de l’IgE-réactivité entre LTP des Rosacées qu’entre ces dernières et rCor a 8  ;

Les résultats de Flinterman et coll. sont donc très importants, tant sur le plan de l’épidémiologie (réactivité vis à vis des LTP ailleurs que dans le Sud) que sur la stratégie à adopter pour développer des tests diagnostics nouveaux.

Les allergènes recombinants "représentatifs" d’une famille moléculaire, comme rPru p 3 pour les LTP, ne sont pas toujours les moyens les plus pertinents pour caractériser une réactivité vis à vis de tous les membres de cette famille. Des facteurs locaux peuvent aussi jouer.

Plus la variété des allergènes testables sera grande et plus les moyens diagnostiques de l’allergologie moléculaire seront efficaces. Dans l’intervalle, les allergènes "représentatifs" procurent néanmoins une aide précieuse.

Fruits à coque et CCD

(voir aussi : Les CCD)

La présence d’épitopes glucidiques IgE-réactifs dans les extraits de FaC n’a pas été beaucoup étudiée, mais on peut s’attendre à ce que les FaC ne fassent pas exception à la règle, quand cela ne serait que du fait des nombreuses protéines extraites des graines simultanément aux allergènes .

Certains allergènes sont glycosylés : la conglutine gamma de l’amande , la légumine de la noix de coco , ainsi que Cor a 11, une viciline de noisette .

Plus globalement, les extraits contenant aussi d’autres protéines que des allergènes, la connaissance des glycomes peut apporter des renseignements utiles  : Wilson a montré que l’amande et la pistache possédaient majoritairement des chaînes glucidiques avec xylose et fucose, contrairement à la noix de coco qui n’a pas de fucose ; la noisette possède un peu de chaînes Lewis a, alors que ces dernières sont majoritaires dans le cas de la noix .

La réalité d’une réactivité de type CCD a été montrée par immuno-capture pour la noix, la noisette et l’amande .

[1] - Ferrer A, Peris A, Giner A, Andreu C, Ligero E, Sempere JM, et al. Oral allergy syndrome by tiger nut. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1021
Tiger nut (Cyperus esculentus) is a tuber belonging to the Cyperaceae family, used in Spain to prepare a milky cold drink called ''horchata'' or tiger nut milk (TM). Few allergic reactions to TM been described in spite of its wide consumption. TM is obtained by maceration in water and filtration. When produced at an industrial level is heat sterilized (UHT-140°C, 3-4 seconds) and canned. Patients and Methods: Patient 1: A 15 year-old rhinitic woman. After consumption of some sips of TM developed OAS -lip and throat angioedema and itching-. On clinical evaluation multiple sensitivities to pneumoallergens and vegetable foods. Patient 2: A 20 year-old male with sensitisation to epithelia, vegetables and fruits. The patient developed lip and tongue oedema with oropharingeal itching after consumption of home-made TM but tolerated UHT-TM. Patient 3: A 27 year-old male with perennial asthma and rhinitis by mite and epithelia. The patient was allergic to shrimp, and developed OAS by fresh TM with tolerance to UHT-TM. A study on the sensitization to TM included SPT to hydrated tiger nut, fresh and UHT TM and tiger nut extract; oral challenge with home-made and UHT-TM (patients 1 and 3), and specific IgE and immunoblotting. Results: SPT with ''fresh'' home-made TM was positive in all three patients. In contrast UHT TM was negative. Oral challenge with TM was done in patients 1 and 3, and resulted positive to fresh TM and negative to UHT TM. Specific IgE to tiger nut was detected in the sera of the three patients ranging from 0.64 to 4.2 kU/l. On immunoblotting IgE bound a 15.7 kDa allergen, with a second band of 30.5 kDa (Patient 3). Conclusions: TM is a potential inducer of OAS but ''ultra-high-temperature'' treatment (UHT) greatly reduces its allergenicity. All the patients tolerated industrial heat- sterilized TM and reacted to ''fresh'' home made TM. Coincident with this finding, prick test with tiger nut extract and TM gave contradictory results, suggesting denaturation of relevant allergens.
[2] - Teuber SS, Comstock SS, Sathe SK, Roux KH. Tree nut allergy. Curr Allergy Asthma Rep 2003;3:54-61
Tree nuts are clinically associated with severe immunoglobulin E-mediated systemic allergic reactions independent of pollen allergy and with reactions that are usually confined to the oral mucosa in patients with immunoglobulin E directed toward cross-reacting pollen allergens. The latter reactions can progress to severe and life-threatening episodes in some patients. Many patients with severe tree nut allergy are co-sensitized to peanut. Clinical studies on cross-reactivity between the tree nuts are few in number, but based on reports to date, avoidance of the other tree nuts once sensitivity is diagnosed appears prudent unless specific challenges are performed to ensure clinical tolerance. Even then, great care must be taken to avoid cross-contamination. As with other severe food allergies, a recurrent problem in clinical management is the failure of physicians to prescribe self-injectable epinephrine to patients who are at risk of anaphylaxis.
[3] - Roux KH, Teuber SS, Sathe SK. Tree Nut Allergens. Int Arch Allergy Immunol 2003;131:234-244
Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.
[4] - Jacquenet S, Moneret-Vautrin DA. Les allergènes de l'arachide et des fruits à coque. Rev Fr Allergol Immunol Clin 2007;47:487-491
La mise au point de nouvelles techniques de désensibilisation utilisant des produits issus de la biologie moléculaire nécessite le contrôle de leur efficacité par des essais cliniques appropriés. Un aperçu des principales molécules proposées par les recherches fondamentales au cours des dernières années est délivré : allergènes recombinants correspondant à des molécules d'allergènes pertinents du fait de leur forte prévalence de sensibilisation, dérivés hypoallergéniques d'allergènes recombinants (polymères, fragments, molécules avec des mutations ou des délétions d'acides aminés), molécules hybrides, épitopes T, épitopes B, molécules conjuguées à des CpG ou d'autres co-stimulateurs. L'activité biologique d'un grand nombre de ces molécules a été démontrée in vivo. La sélection de molécules candidates pour l'immunothérapie a d'abord comporté des études préliminaires effectuées en ouvert sur un nombre restreint de patients. Les auteurs rapportent ici les résultats de cinq études de désensibilisations effectuées par injection, réalisées en double insu, le groupe témoin étant un placebo et/ou un groupe traité par l'extrait naturel (dans deux études). Les résultats cliniques les plus significatifs sur le plan statistique ont été observés avec des allergènes recombinants non modifiés, sans survenue d'accidents systémiques sévères. Dans les cinq études, on relève une augmentation importante des IgG spécifiques vis-à-vis de l'allergène naturel et vis-à-vis des allergènes recombinants. La réduction des tests cutanés dans les groupes traités par rapport au groupe placebo est significative dans deux études sur trois. Des études comportant un nombre de patients plus important sont maintenant souhaitables pour répondre aux exigences réglementaires du développement clinique des allergènes recombinants.
[5] - Kewalramani A, Maleki SJ, Cheng H, Teuber S. Cross-Reactivity Among Almond, Peanut and Other Tree Nuts in Almond Allergic Patients. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°127
RATIONALE: Almond is known to be cross-reactive with other members of the Rosaceae family, but little has been reported on its cross reactivity with other seeds and tree nuts. We assessed cross-reactivity of raw and roasted almond, peanut, and other raw and roasted tree nuts METHODS: IgE immunoblotting was performed using sera from three patients with a history of almond allergy and one patient with unknown history, but an elevated specific IgE (Pharmacia ImmunoCap). Samples included raw and roasted almond, apricot seed, sunflower seed, pine nut, walnut and pecan. The samples were all normalized according to protein content and subjected to SDS-PAGE and then Western blot analysis with sera from all patients. Additionally, antibodies to Ara h 1, Ara h 2, and Ara h 3 were used to identify vicilin, glycinin, and conglutin homologues RESULTS: Immunoblot results showed extensive IgE cross-reactivity between the almond and apricot seed samples. However, extensive crossreactivity seemed to exist with sunflower and pine nut in all patients. In most patients, cross-reactivity seemed to also be present with the walnut and pecan samples CONCLUSIONS: As expected, extensive IgE cross-reactivity was seen between almond and apricot seed as both are members of the Rosaceae family and Prunoideae subfamily. However, previously unidentified cross-reactive proteins may exist with sunflower, pine nut, walnut, and pecan as well.
[7] - Vega A, Dominguez C, Cosmes P, Martinez A, Bartolome B, Martinez J, et al. Anaphylactic reaction to ingestion of Quercus ilex acorn nut. Clin Exp Allergy 1998;28:739-742
A patient experienced an anaphylactic reaction after eating acorn nuts, fruit of the holm oak (Quercus ilex), one of the most abundant trees in Spain. Several urticaria episodes upon ingestion of peanuts were also referred. OBJECTIVE: To assess the hypersensitivity reaction to acorn and to characterize the allergenic proteins involved. METHODS: Cutaneous tests were performed using the skin-prick technique, using a large variety of grass, tree and weed pollens as well as fresh nuts and nut extracts. Specific IgE determination was assessed by RAST. IgE binding bands were determined by SDS-PAGE immunoblotting. RESULTS: Skin-prick tests were strongly positive with acorn and peanut. Olea europaea, Quercus alba, Quercus ilex and grass pollens also elicited a weal higher than negative control. Patient serum had measurable levels of IgE antibodies especially to acorn, peanut and grass pollens. Only one protein band, of 17.9 kDa molecular mass, showed IgE-binding properties in the acorn extract. The possible homology of this strong allergenic protein with the group 1 tree pollen allergens was evidenced by the partial inhibition of the western blot with Bet v 1. CONCLUSIONS: We present a case of anaphylaxis to acorn ingestion as demonstrated by in vivo and in vitro results. A 17.9 kDa IgE-binding band, showing some homology to group 1 pollen tree allergens, was recognized by patient serum.
[8] - Roux KH, Teuber SS, Sathe SK. Tree Nut Allergens. Int Arch Allergy Immunol 2003;131:234-244
Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.
[10] - Pumphrey RSH, Farragher A, Summers C. Patterns of fatal and non-fatal reactions to different types of nuts. BSACI Annual Conference, Nottingham, June 30th-July 2nd, 2003, Abstract 13
Background The prevalence of nut allergy is increasing; its diagnosis causes anxiety in patients and their families through fear of a fatal reaction. Our previous statistics suggest that not all nuts are equally dangerous. Objectives To compare the clinical characteristics of fatal and non-fatal reactions to a range of nuts. Methods Data for fatal nut reactions were taken from a register of all fatal anaphylactic reactions in the UK, 1992ˆ2002. A pro forma history was taken from all patients referred following suspected allergic reactions to nuts. Details of exposure, timing and severity of symptoms were obtained for at least their first, worst and most recent reactions. Results Over 2000 non-fatal reactions were studied, due to peanuts (41%), walnut (3%), brazil nut (4%), almond (4%), hazelnut (3%), pistachio (2%), cashew (4%), mixed or unidenti?ed nuts (40%). Only 34 fatal reactions to nuts were identi?ed, due to peanuts (29%), walnuts (15%), brazil nuts (9%), almonds (6%), hazelnut (3%), mixed or unidenti?ed nuts (35%). There were differences in the severity of each type of symptom studied between the different nuts ˆ for example, compared with peanuts, brazil nuts caused more severe reactions with more dominant throat and upper airway symptoms. Discussion In addition to the amount of causative allergen eaten, circumstances of the reaction and pre-existing pathology, the speci?city of the nut allergy may also be important in determining the severity of the reaction.
[11] - MacDougall CF, Cant AJ, Colver AF. How dangerous is food allergy in childhood ? The incidence of severe and fatal allergic reactions across the UK and Ireland. Arch Dis Child 2002;86:236-239
AIMS: To discover the incidence of fatal and severe allergic reactions to food in a large population of children. METHODS: A retrospective search for fatalities in children 0-15 years from 1990 to February 1998, primarily of death certification at offices of national statistics. A prospective survey of fatal and severe reactions from March 1998 to February 2000, primarily through the British Paediatric Surveillance Unit. MAIN OUTCOME MEASURES: were deaths and severe reactions. A case was deemed severe if one or more of the following criteria was met: cardiorespiratory arrest; need for inotropic support; fluid bolus >20 ml/kg; more than one dose of epinephrine; more than one dose of nebulised bronchodilator. A case was deemed near fatal if intubation was necessary. RESULTS: The UK under 16 population is 13 million. Over the past 10 years, eight children died (incidence of 0.006 deaths per 100 000 children 0-15 years per year). Milk caused four of the deaths. No child under 13 died from peanut allergy. Two children died despite receiving early epinephrine before admission to hospital; one child with a mild food allergic reaction died from epinephrine overdose. Over the past two years, there were six near fatal reactions (none caused by peanut) and 49 severe ones (10 caused by peanut), yielding incidences of 0.02 and 0.19 per 100 000 children 0-15 years per year respectively. Coexisting asthma is more strongly associated with a severe reaction than the severity of previous reactions. CONCLUSIONS: If 5% of the child population have food allergy, the risk that a food allergic child will die from a food allergic reaction is about 1 in 800 000 per year. The food allergic child with asthma may be at higher risk. Prescribing an epinephrine autoinjector requires a careful balance of advantages and disadvantages.
[12] - Bock SA, Muñoz-Furlong A, Sampson HA. Fatalities due to anaphylactic reactions to foods. J Allergy Clin Immunol 2001;107:191-193
Fatal anaphylactic reactions to foods are continuing to occur, and better characterization might lead to better prevention. The objective of this report is to document the ongoing deaths and characterize these fatalities. We analyzed 32 fatal cases reported to a national registry, which was established by the American Academy of Allergy, Asthma, and Immunology, with the assistance of the Food Allergy and Anaphylaxis Network, and for which adequate data could be collected. Data were collected from multiple sources including a structured questionnaire, which was used to determine the cause of death and associated factors. The 32 individuals could be divided into 2 groups. Group 1 had sufficient data to identify peanut as the responsible food in 14 (67%) and tree nuts in 7 (33%) of cases. In group 2 subjects, 6 (55%) of the fatalities were probably due to peanut, 3 (27%) to tree nuts, and the other 2 cases were probably due to milk and fish (1 [9%] each). The sexes were equally affected; most victims were adolescents or young adults, and all but 1 subject were known to have food allergy before the fatal event. In those subjects for whom data were available, all but 1 was known to have asthma, and most of these individuals did not have epinephrine available at the time of their fatal reaction. Fatalities due to ingestion of allergenic foods in susceptible individuals remain a major health problem. In this series, peanuts and tree nuts accounted for more than 90% of the fatalities. Improved education of the profession, allergic individuals, and the public will be necessary to stop these tragedies.
[13] - Rancé F, Bidat E, Bourrier T, Sabouraud D. Cashew allergy: observations of 42 children without associated peanut allergy. Allergy 2003;58:1311-1314
BACKGROUND: Cashew allergy seems to be increasingly frequent. The goal of the present study was to analyse the clinical features and results of investigations of 42 children with cashew allergy . METHODS: The clinical features and results of skin prick tests, specific IgE assays, and food challenges were analysed . RESULTS: The mean age at first allergic reaction was 2 years and the mean age at diagnosis of cashew allergy was 2.7 years. One in five children (12%) had a prior history of exposure to cashew nuts. Fifty-six per cent had skin symptoms, 25% had respiratory signs and 17% had digestive signs. Eighteen children had proven, associated food allergies (pistachio, seven; egg, five; mustard, three; shrimp, two; cow milk, one). The mean wheal diameter of the skin prick tests was 7 mm (3-16 mm) and the mean specific IgE level was 3.1 kUA/L (<0.35->100 kUA/L). Eight children had positive food challenges . CONCLUSION: The increase in cashew allergy is worrying because it affects young children who may have a reaction without ever having been exposed to cashews. Almost one-third of children are allergic to pistachios, which belong to the same botanical family as cashews. Clinical history is generally and sufficiently suggestive to diagnose cashew allergy without recourse to food challenges.
[15] - Wensing M, Penninks AH, Hefle SL, Akkerdaas JH, van Ree R, Koppelman SJ, et al. The range of minimum provoking doses in hazelnut-allergic patients as determined by double-blind, placebo-controlled food challenges. Clin Exp Allergy 2002;32:1757-1762
Background The risk for allergic reactions depends on the sensitivity of individuals and the quantities of offending food ingested. The sensitivity varies among allergic individuals, as does the threshold dose of a food allergen capable of inducing an allergic reaction. Objective : This study aimed at determining the distribution of minimum provoking doses of hazelnut in a hazelnut-allergic population. Method s : Thirty-one patients with a history of hazelnut-related allergic symptoms, a positive skin prick test to hazelnut and/or an elevated specific IgE level, were included. Double-blind, placebo-controlled food challenges (DBPCFC) were performed with seven increasing doses of dried hazelnut (1 mg to 1 g hazelnut protein) randomly interspersed with seven placebo doses. Result s : Twenty-nine patients had a positive challenge. Itching of the oral cavity and/or lips was the first symptom in all cases. Additional gastrointestinal symptoms were reported in five patients and difficulty in swallowing in one patient. Lip swelling was observed in two patients, followed by generalized urticaria in one of these. Threshold doses for eliciting subjective reactions varied from a dose of 1 mg up to 100 mg hazelnut protein (equivalent to 6.4-640 mg hazelnut meal). Extrapolation of the dose-response curve showed that 50% of our hazelnut-allergic population will suffer from an allergic reaction after ingestion of 6 mg (95% CI, 2-11 mg) of hazelnut protein. Objective symptoms were observed in two patients after 1 and 1000 mg, respectively. Conclusion : DBPCFCs demonstrated threshold doses in half of the hazelnut-allergic patients similar to doses previously described to be hidden in consumer products. This stresses the need for careful labelling and strategies to prevent and detect contamination of food products with hazelnut residues.
[17] - Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-1045
BACKGROUND: Peanut and tree nut allergy are common, increasing in prevalence and the commonest food cause of anaphylaxis. In the USA, 7.8% are sensitized (have nut-specific IgE), but not all those sensitized are allergic. Lack of data makes interpretation of tests for nut-specific IgE difficult . OBJECTIVES: This is the first study to investigate the clinical significance of test results for peanut and tree nut allergy in allergic or tolerant patients. Findings are related to the severity of the allergy . METHOD: An observational study of 1000 children and adults allergic to at least one nut. History of reactions (severity graded) or tolerance to up to five nuts was obtained and skin prick test (SPT)/serum-specific IgE (CAP) performed . RESULTS: There was no correlation between SPT size and graded severity of worst reaction for all nuts combined or for peanut, hazelnut, almond and walnut. For CAP, there was no correlation for all nuts. Where patients tolerated a nut, 43% had positive SPT of 3-7 mm and 3% > or = 8 mm. For CAP, 35% were positive (0.35-14.99 kU/L) and 5% > or = 15 kU/L. In SPT range 3-7 mm, 54% were allergic and 46% were tolerant. There was poor concordance between SPT and CAP (66%). Of patients with a clear nut-allergic history, only 0.5% had negative SPT, but 22% negative CAP . CONCLUSIONS: Magnitude of SPT or CAP does not predict clinical severity, with no difference between minor urticaria and anaphylaxis. SPT is more reliable than CAP in confirming allergy. Forty-six per cent of those tolerant to a nut have positive tests > or = 3 mm (sensitized but not allergic). One cannot predict clinical reactivity from results in a wide 'grey area' of SPT 3-7 mm; 22% of negative CAPs are falsely reassuring and 40% of positive CAPs are misleading. This emphasizes the importance of the history. Understanding this is essential for accurate diagnosis. Patients with SPT > or = 8 mm and CAP > or = 15 kU/L were rarely tolerant so these levels are almost always (in > or = 95%) diagnostic. During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.
[19] - Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-1045
BACKGROUND: Peanut and tree nut allergy are common, increasing in prevalence and the commonest food cause of anaphylaxis. In the USA, 7.8% are sensitized (have nut-specific IgE), but not all those sensitized are allergic. Lack of data makes interpretation of tests for nut-specific IgE difficult . OBJECTIVES: This is the first study to investigate the clinical significance of test results for peanut and tree nut allergy in allergic or tolerant patients. Findings are related to the severity of the allergy . METHOD: An observational study of 1000 children and adults allergic to at least one nut. History of reactions (severity graded) or tolerance to up to five nuts was obtained and skin prick test (SPT)/serum-specific IgE (CAP) performed . RESULTS: There was no correlation between SPT size and graded severity of worst reaction for all nuts combined or for peanut, hazelnut, almond and walnut. For CAP, there was no correlation for all nuts. Where patients tolerated a nut, 43% had positive SPT of 3-7 mm and 3% > or = 8 mm. For CAP, 35% were positive (0.35-14.99 kU/L) and 5% > or = 15 kU/L. In SPT range 3-7 mm, 54% were allergic and 46% were tolerant. There was poor concordance between SPT and CAP (66%). Of patients with a clear nut-allergic history, only 0.5% had negative SPT, but 22% negative CAP . CONCLUSIONS: Magnitude of SPT or CAP does not predict clinical severity, with no difference between minor urticaria and anaphylaxis. SPT is more reliable than CAP in confirming allergy. Forty-six per cent of those tolerant to a nut have positive tests > or = 3 mm (sensitized but not allergic). One cannot predict clinical reactivity from results in a wide 'grey area' of SPT 3-7 mm; 22% of negative CAPs are falsely reassuring and 40% of positive CAPs are misleading. This emphasizes the importance of the history. Understanding this is essential for accurate diagnosis. Patients with SPT > or = 8 mm and CAP > or = 15 kU/L were rarely tolerant so these levels are almost always (in > or = 95%) diagnostic. During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.
[20] - Clark AT, Ewan PW. The development and progression of allergy to multiple nuts at different ages. Pediatr Allergy Immunol 2005;16:507-511
The aims of this study were to define the development of sensitization and clinical allergy to multiple nut types by age, and to identify associations. This observational cross- sectional study was carried out in a tertiary level allergy clinic. Seven hundred and eighty four nut-allergic children, prospectively enrolled on first attendance with history of a type-1 allergic reaction shortly after definite nut ingestion with evidence of sensitization (presence of nut-specific IgE) by skin prick test (SPT) to peanut, Brazil, almond, hazel and walnut (positive > or = 3 mm). Details of exposure (tolerance or allergy) to each nut were obtained. The main outcome measures were mono or multi-sensitization (specific IgE to one or more than one nut type, demonstrated by SPT); mono or multi-allergy (clinical allergy to one or more than one nut type). By 2 yr of age at least 19% were multi-sensitized, and 2% multi-allergic. Increasing proportions were exposed to multiple nut types with increasing age (23% at 2 yr to 73% by 10 yr) and greater proportions were multi-sensitized (19% at 2 yr to 86% at 5-14 yr) and multi-allergic (2% at 2 yr to 47% at 14 yr). This study is the first to define the natural history of multiple nut allergies in childhood. New findings are that a large proportion of those aged 0-1 yr with nut allergy are already sensitized (have specific IgE) to multiple nut types, implying in utero or early life sensitization; those who present later in childhood are increasingly likely to be sensitized and clinically allergic to multiple nuts. This is related to increased duration of allergy and exposure to multiple nut types with age. Children with nut allergy should avoid all nut types from the onset.
[21] - Ewan PW, Clark AT. Long-term prospective observational study of patients with peanut and nut allergy after participation in a management plan. Lancet 2001;357:111-115
BACKGROUND: Peanut and nut allergy is common and the most frequent cause of severe or fatal reactions to foods. Current advice is poor--doctors give an epinephrine injector to patients, without training or advice on nut avoidance--so that further reactions are common and deaths occur. We devised and assessed a management programme providing advice on nut avoidance and emergency medication. METHODS: Unselected referrals with confirmed peanut or tree-nut allergy were recruited. Severity of nut allergy was graded 1-5 and emergency medication allocated accordingly: oral antihistamine with or without inhaled or injected epinephrine. Patients, parents, and school staff received verbal and written advice on nut avoidance as well as training in recognition and self-treatment of reactions, with a written treatment plan. At follow-up (more than 13610 patient months) retraining was given and details of further reactions obtained. FINDINGS: 88 (15%) of 567 patients had a follow-up reaction of reduced severity. 62 of 88 were mild (grades 1-3, mainly cutaneous) and 49 patients used oral antihistamine, six inhaled adrenaline, and ten took no treatment. 12 of 12 patients with a moderate follow-up reaction improved after inhaled epinephrine. Only three (0.5%) of 567 patients, aged 27-40 years, had a severe follow-up reaction (involving dyspnoea) compared with 12% initially. Only one of 567 changed from a mild index reaction to a severe follow-up reaction. Patients with a moderate/severe (grade 4-5) reaction were older (median 18 years vs 9 years; p=0.03) and nine of 26 received injected epinephrine which was always effective. 85% of patients had no further reactions. Severity was related to the amount of nut eaten. INTERPRETATION: Self-treatment was effective (inhaled epinephrine for early laryngeal oedema and an epinephrine injector for severe reactions) but provision of this treatment, including who should carry epinephrine, required assessment of allergy severity. Our management plan was effective, and our results indicate that patients should be referred to specialist allergy centres for advice on nut avoidance.
[22] - Asero R, Mistrello G, Roncarolo D, Amato S, Caldironi G, Barocci F, et al. Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study. Allergy 2002;57:900-906
BACKGROUND: Lipid transfer proteins (LTP) are highly conserved and widely distributed throughout the plant kingdom. Recent studies demonstrated immunological cross-reactivity between LTP from many botanically unrelated fruits and vegetables and concluded that LTP are pan-allergens. This study aimed to evaluate the clinical relevance of such cross-reactivity in a group of subjects monosensitized to LTP . METHODS: Twenty LTP-hypersensitive patients were selected from a population of about 600 subjects with history of Rosaceae allergy by means of: 1) negative skin prick test (SPT) with a commercial birch pollen extract; 2) positive SPT with a commercial plum extract, rich in LTP but virtually lacking both Bet v 1-like proteins and profilin; 3) in-vitro IgE reactivity to the 9-10 kDa fraction of peach peel or immunoblot with peach peel showing a single band at 10 kDa; and 4) total inhibition of reactivity to whole peach extract (containing Bet v 1-related allergen, profilin, and LTP) by purified peach LTP on enzyme-linked immunoassay (ELISA). Allergy to foods other than Rosaceae was ascertained by careful interview and analysis of medical recordings. SPT with a large series of plant-derived foods were carried out as well. The cross reactivity between LTPs from botanically unrelated plant-derived foods was assessed by ELISA inhibition tests using walnut and peanut extracts as substrate, and peach LTP as inhibitor . RESULTS: All patients reported allergic reactions after the ingestion of at least one from a large number of vegetable foods other than Rosaceae, and in several cases clinical reactions were very severe (anaphylaxis, asthma, urticaria/angioedema). Nuts and peanuts were the most frequently reported causes of allergic reactions (80% and 40% of patients, respectively). All patients showed positive SPT to several non-Rosaceae food extracts. SPT with nuts, peanut, legumes, celery, rice, and corn were positive in the majority of patients. In ELISA inhibition studies, absorption of sera with peach LTP caused complete inhibition of IgE reactivity to walnut and peanut in all cases . CONCLUSION: LTP is a clinically relevant pan-allergen. Most Rosaceae-allergic, LTP-hypersensitive patients experience adverse reactions after ingestion of botanically unrelated plant-derived foods as well. In view of the high prevalence and severity of the allergic reactions induced, hazelnut, walnut, and peanut should be regarded as potentially hazardous for these patients.
[23] - Teuber SS, Pastorello E, Peterson WR, Dandekar AM, Uratsu S. Cloning and Characterization of Walnut Lipid Transfer Protein: Not a Major Allergen in Walnut-Allergic U.S. Patients. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°172
RATIONALE: Lipid transfer protein, a protein with inherent resistance to gastric digestion, has been isolated from walnut kernel and determined to bind IgE from Italian patients with walnut allergy. Many of these patients had been initially sensitized to lipid transfer protein in peaches We sought to determine if lipid transfer protein was also a major allergen for U.S. patients with severe walnut allergy, and if the cloned, expressed protein could inhibit IgE binding to the native protein METHODS: RNA was isolated from walnut embryos and used as a template for cDNA. 3' and 5' RACE was performed using primers based on the coding sequence for walnut lipid transfer protein from a walnut embryo EST database. Full-length cDNA was subcloned into the pMALc2X vector and expressed as a recombinant fusion protein in E. coli Native lipid transfer protein, sera from Italian patients, and 34 sera from U.S. patients, most of whom had severe, life-threatening reactions to walnut, were used in IgE immunoblotting and inhibitions RESULTS: The sera from Italian patients with walnut allergy and IgE towards lipid transfer protein were able to bind the recombinant, and the recombinant was able to absorb out reactivity to the native walnut lipid transfer protein and vice-versa. However, sera from U.S. patients, except one, failed to show IgE binding to the native or recombinant lipid transfer protein CONCLUSIONS: Although lipid transfer proteins are an important class of food allergens, it appears that there are dramatic geographic influences on sensitization to this class of allergen which warrant further investigation Funding: Food Allergy Initiative
[24] - Rodriguez J, Poza P, Crespo JF. Follow-Up of Immunological Reactivity in 27 Patients Allergic to Nuts and Seeds. J Allergy Clin Immunol 2005;115(2 suppl.):S94
RATIONALE: There is limited information on the natural course of patients with positive skin testing and/or CAP FEIA to peanuts and/or tree nuts, but clinical tolerance. We evaluated if immunological reactivity to nuts developed into clinical reactivity over time METHODS: Twenty-seven patients (median= 25 yr.) with proven clinical allergy (DBPCFC procedure) to nuts [almond (AL), 3 patients; hazelnut (HN), 13; peanut (PN), 6; chestnut (ChN), 8; walnut (WN), 12; pistachio (P), 2; and sunflower seed (S), 6] were included in the study. Patients underwent skin prick testing and specific IgE determinations (CAP-FEIA) to other nuts with proven tolerance and, if positive, new clinical reactivity to nuts was assessed by phone interview, followed by oral provocations Median follow-up (months) was: AL, 51.5; HZ, 49.5; PN, 43; ChN, 38; WN, 65.5; P, 44.5; and S, 42 RESULTS: A total of 89 immunologic reactivities to nuts were detected (17 to AL, 7 to HZ, 17 to PN, 16 to ChN, 12 to WN, 10 to P, and 11 to S) After the follow- up period, 24 patients (80 immunologic reactions) could be contacted by telephone (89% participants). They reported intentional avoidance of 26 nuts (33%); ingestion with tolerance of 53 nuts (66%), and eating with symptoms to 1 nut (WN) (1%). A further clinical evaluation including DBPCFC confirmed one new case of clinical allergy to WN CONCLUSIONS: Patients clinically reactive to a nut have also an extensive immunologic reactivity to other nuts, which could be safely consumed, as new cases of clinical allergy seem to be infrequent
[25] - Fleischer DM, Conover-Walker MK, Matsui EC, Wood RA. The natural history of tree nut allergy. J Allergy Clin Immunol 2005;116:1087-1093
BACKGROUND: Although 20% of children outgrow peanut allergy, the natural history of tree nut (TN) allergy has not been well studied . OBJECTIVE: The goals of the study were to estimate the proportion of children who outgrow TN allergy and examine predictors of outgrowing it . METHODS: Patients with TN allergy, defined as a history of reaction on ingestion and evidence of TN-specific IgE (TN-IgE) or positive TN-specific IgE level but no history of ingestion, were evaluated. If all current TN-IgE levels were less than 10 kilounits of antibody (kU(A))/L, double-blind, placebo-controlled food challenges were offered. Patients who had undergone open TN challenges as part of routine clinical care were also included . RESULTS: Two hundred seventy-eight patients with TN allergy were identified. One hundred one (36%) had a history of acute reactions, 12 (12%) of whom had reactions to multiple TNs and 73 (63%) of whom had a history of moderate-to-severe reactions. Nine of 20 patients who had previously reacted to a TN passed challenges, so that 9 (8.9%; 95% CI, 4% to 16%) of 101 patients with a history of prior TN reactions outgrew TN allergy. Fourteen of 19 who had never ingested TNs but had detectable TN-specific IgE levels passed challenges. One hundred sixty-one did not meet the challenge criteria, and 78 met the criteria but declined challenges. Looking at specific TN-IgE cutoffs, 58% with TN-IgE levels of 5 kU(A)/L or less and 63% with TN-IgE levels of 2 kU(A)/L or less passed challenges . CONCLUSIONS: Approximately 9% of patients outgrow TN allergy, including some who had prior severe reactions. Although ideal cutoffs for challenge cannot be firmly recommended on the basis of these data, patients aged 4 years or older with all TN-IgE levels of 5 kU(A)/L or less should be considered for challenge.
[26] - 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.
[27] - Sanchez C, Frémont S. Conséquences des traitements thermiques et de la formulation sur la structure et l'allergénicité des protéines alimentaires. Rev Fr Allergol Immunol Clin 2003;43:13-20
Une augmentation importante du nombre de cas d'allergies alimentaires a été constatée au cours des cinq dernières années. Parmi les causes possibles, les procédés alimentaires et les interactions entre ingrédients et additifs sont fortement suspectés. Cette revue présente l'état des connaissances sur la relation entre chauffage, formulation, structure et allergénicité des protéines. Les procédés de fabrication alimentaire comprennent un ensemble d'opérations unitaires thermiques et mécaniques dont l'objet est de structurer, texturer et permettre une conservation satisfaisante de l'aliment. Les opérations de chauffage entraînent dans la plupart des cas une dénaturation irréversible de la conformation de la protéine pouvant conduire à son agrégation. Ces changements structuraux ne sont pas corrélés avec une diminution du potentiel allergénique des protéines. Selon les cas, un chauffage peut n'avoir aucun effet, diminuer ou augmenter ce potentiel. L'existence d'épitopes séquentiels et conformationnels, le démasquage de nouveaux épitopes ou la modification d'épitopes par réaction de Maillard peut expliquer partiellement les résultats reportés dans la littérature. Des interactions entre protéines allergènes et autres molécules présentes dans les aliments peuvent entraîner des modifications conformationnelles des protéines, même sans chauffage, et affecter leur stabilité thermique. En particulier, une augmentation ou une diminution de l'agrégation thermique des protéines peuvent être observées. L'effet de ces interactions sur le potentiel allergénique des protéines est aujourd'hui pratiquement inconnu.
[29] - Maleki SJ. Food processing: effects on allergenicity. Curr Opin Allergy Clin Immunol 2004;4:241-245
PURPOSE OF REVIEW: There has been an increase in the prevalence of food allergy, asthma and severe reactions to foods in the past decade. The reason for this increase is unknown and despite the potential for a lethal outcome, no treatments or therapies are available. Many areas are being explored to understand the increase in food allergy and the role of modern processing techniques is one of them. RECENT FINDINGS: The fact that processing influences allergenicity is not a new concept, but due to some definitive studies in the last few years the topic has been revitalized. Not only is the processed form of a particular allergen important in assessment of allergenicity, but the role of the food matrix in which an allergen is processed must also be evaluated. The issue of allergenicity of particular foods is therefore highly complex, and even with our existing knowledge we have just begun to understand some of the contributions of processing to allergenic properties of proteins and foods. SUMMARY: Researchers and medical personnel have begun to weigh the benefits and potential disadvantages of assessing the allergenicity of foods in unprocessed forms. In this review, the known effects of processing on the allergenicity of various foods will be discussed along with the implications of the most recent studies on the future direction of research in this area.
[31] - Schöll I, Untersmayr E, Bakos N, Roth-Walter F, Gleiss A, Boltz-Nitulescu G, et al. Antiulcer drugs promote oral sensitization and hypersensitivity to hazelnut allergens in BALB/c mice and humans. Am J Clin Nutr 2005;81:154-160
BACKGROUND: Hazelnut allergy can be a consequence of sensitization to cross-reactive pollen, especially from the Fagales family. However, severe allergic reactions after ingestion of hazelnuts without associated pollen allergy have been reported. In these cases, oral sensitization by hazelnut ingestion is plausible. OBJECTIVE: We have reported that antiulcer drugs promote oral sensitization to digestion-labile food allergens. Because hazelnut proteins were sensitive to gastric digestion in our in vitro assay, we aimed to analyze the effect of antiulcer treatment on oral sensitization to hazelnut proteins. DESIGN: BALB/c mice were fed hazelnut extract with or without antiulcer drugs. In parallel, gastroenterologic patients (n = 153) were screened during antiulcer treatment for specific immunoglobulin (Ig) E to hazelnut and inhalative allergens in vitro and in vivo. RESULTS: Mice fed hazelnut extract in combination with antiulcer drugs formed anaphylactogenic IgG1 toward hazelnut and developed type I skin reactivity to hazelnut extract. In the human study population, 5 of 153 (3.3%) patients developed hazelnut-specific IgE, 4 of 5 developed specific skin reactivity, 3 of 5 had a positive result to oral provocation, and 2 of 5 manifested a food allergy to hazelnut after a 3-mo course of antiulcer treatment. Immunoblot testing with recombinant allergens showed that hazelnut, but not Fagales pollen, was the genuine elicitor in mice and humans. CONCLUSION: Our experimental and epidemiologic data suggest that the intake of antiulcer drugs may lead to the induction of immediate-type food hypersensitivity toward hazelnut.
[32] - Schimek EM, Zwölfer B, Briza P, Jahn-Schmid B, Vogel L, Vieths S, et al. Gastrointestinal digestion of Bet v 1-homologous food allergens destroys their mediator-releasing, but not T cell–activating, capacity. J Allergy Clin Immunol 2005;116:1327-1333
BACKGROUND: Food allergy to apples, hazelnuts, and celery is frequent in individuals with birch pollen allergy because IgE antibodies specific for the major birch pollen allergen, Bet v 1, cross-react with structurally related allergens in these foods. In addition, T lymphocytes specific for Bet v 1 also cross-react with these dietary proteins . OBJECTIVE: We sought to evaluate the effects of simulated gastrointestinal degradation of Bet v 1-related food allergens on their mediator-releasing and T cell-activating capacity . METHODS: Recombinant Mal d 1, Cor a 1.04, and Api g 1 were incubated separately with pepsin and trypsin. Binding of IgE was tested in immunoblots. After successive incubation with both enzymes, allergens were tested in mast cell mediator release assays and used to stimulate PBMCs and Bet v 1-specific T-cell lines and clones. Proteolytic fragments of allergens were analyzed and sequenced by means of mass spectrometry . RESULTS: Pepsin completely destroyed IgE binding of all allergens within 1 second, and trypsin completely destroyed IgE binding of all allergens within 15 minutes, except for the major hazelnut allergen, which remained intact for 2 hours of trypsinolysis. Allergens after gastrointestinal digestion did not induce basophil activation but induced proliferation in PBMCs from allergic and nonallergic individuals. Digested Mal d 1 and Cor a 1.04 still activated Bet v 1-specific T cells, whereas digested Api g 1 did not. Different proteolytic fragments of Mal d 1 and Cor a 1.04 matching relevant Bet v 1 T-cell epitopes were found . CONCLUSION: Gastrointestinal degradation of Bet v 1-related food allergens destroys their histamine-releasing, but not T cell-activating, property. Our data emphasize that birch pollen-related foods are relevant activators of pollen-specific T cells.
[33] - 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
[34] - Fu TJ, Abbott UR, Hatzos C. Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study. J Agric Food Chem 2002;50:7154-7160
Information on the comparative digestibility of food allergens and nonallergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. In this work, we compared the digestive stability of a number of food allergens and proteins of unproven allergenicity and examined whether allergens possess a higher stability than nonallergenic proteins of similar cellular functions, and whether there is a correlation between protein digestibility and allergenicity. The stability of groups of storage proteins, plant lectins, contractile proteins, and enzymes, both allergens and proteins with unproven allergenicity, in a standard simulated gastric fluid and a standard simulated intestinal fluid was measured. Food allergens were not necessarily more resistant to digestion than nonallergenic proteins. There was not a clear relationship between digestibility measured in vitro and protein allergenicity.
[35] - Venkatachalam M, Teuber SS, Roux KH, Sathe SK. Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins. J Agric Food Chem 2002;50:3544-3548
Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart.
[36] - Tawde P, Venkatesh YP, Wang F, Teuber SS, Sathe SK, Roux KH. Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen. J Allergy Clin Immunol 2006;118:915-922
BACKGROUND: The identity of allergenic almond proteins is incomplete . OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen . METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin . RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins . CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.
[38] - Castillo M, Ibero M, Vinas M, Bartolome B. Cross Sensitization between Parietaria judaica Pollen and Vegetal Foods. J Allergy Clin Immunol 2008;121:S184
RATIONALE: Some researchers demonstrated that sensitization to various pollens predispose to suffer from food allergy. We decided to investigate the relation between the sensitization to Parietaria judaica pollen and the subsequent develop of food allergy. METHODS: We recruited 27 patients allergic only or in major degree to Parietaria judaica pollen who underwent a protocol consisting on: cutaneous tests to extracts from Parietaria judaica pollen, artichoke, hazelnut, peanut, pistachio, peach peel and peach pulp; clinical history on adverse reactions with peach, nuts or artichoke and specific IgE to Parietaria judaica pollen, artichoke (flower, stem and leaves), hazelnut, peanut, peach (peel and lipid transfer protein (LTP)) and almond (roasted and raw). Finally we performed SDS-PAGE immunoblotting with Parietaria judaica pollen extract. RESULTS: All patients had serum specific IgE to Parietaria judaica pollen (class 2 to 4) without significant differences in the IgE levels to Par j 1 and Par j 2. Nine from 27 (33.3%) of them had clinical and/or positive tests and/or clinical allergic history to peach, nuts or artichoke. All patient sera revealed the same IgE-binding bands at 16, 29, 40 and 55 kDa; all of these bands turned to be different molecular forms of the major allergens (Par j 1 and Par j 2). CONCLUSIONS: As the main allergens from Parietaria judaica pollen are non-specific LTP, the fact that only some of the P. judaica pollen allergic patients develop food allergy, could be explained in terms of Par j 1, Par j 2 specific IgE concentration, differences on epitope recognition or both mechanisms.
[39] - Untersmayr E, Bakos N, Schöll I, Kundi M, Roth-Walter F, Szalai K, et al. Anti-ulcer drugs promote IgE formation toward dietary antigens in adult patients. FASEB J 2005;19:656-658
Recently, we have demonstrated that anti-ulcer drugs, such as H2-receptor blockers and proton pump inhibitors, promote the development of immediate type food allergy toward digestion-labile proteins in mice. The aim of this study was to examine the allergological relevance of these findings in humans. In an observational cohort study, we screened 152 adult patients from a gastroenterological outpatient clinic with negative case histories for atopy or allergy, who were medicated with H2-receptor blockers or proton pump inhibitors for 3 months. IgE reactivities to food allergens before and after 3 months of anti-acid treatment were compared serologically. Ten percent of the patients showed a boost of preexisting IgE antibodies and 15% de novo IgE formation toward numerous digestion-labile dietary compounds, like milk, potato, celery, carrots, apple, orange, wheat, and rye flour. Thus, the relative risk to develop food-specific IgE after anti-acid therapy was 10.5 (95% confidence interval: 1.44-76.48). The long-term effect was evaluated 5 months after therapy. Food-specific IgE could still be measured in 6% of the patients, as well as significantly elevated serum concentrations of ST2, a Th2-specific marker. An unspecific boost during the pollen season could be excluded, as 50 untreated control patients revealed no changes in their IgE pattern. In line with our previous animal experiments, our data strongly suggest that anti-ulcer treatment primes the development of IgE toward dietary compounds in long-term acid-suppressed patients.
[40] - Müller U, Lüttkopf D, Hoffmann A, Petersen A, Becker WM, Schocker F, et al. Allergens in raw and roasted hazelnuts (Corylus avellana) and their cross-reactivity to pollen. Eur Food Res Technol 2000;212:2-12
Hazelnuts provoke one of the most frequent pollen-associated food allergies. In this study, hazelnut allergens were investigated and immunologically characterized, focussing on their heat stability and cross-reactivity with known allergenic structures. 27 sera from hazelnut-allergic patients and 28 sera from children with positive CAP classes for hazelnut and birch pollen were submitted to immunoblot and immunoblot inhibition. The major hazelnut allergen was found to present a mol. wt. of approx. 17-18 kDa, and to share IgE epitopes with Bet v 1, the major birch pollen allergen. This allergen was recognized by IgE of 93% of hazelnut-allergic patients and by 79% of sensitized children. A 48 kDa glycoprotein was identified as a minor hazelnut allergen with cross-reactive carbohydrate determinants. The major N-glycan species was determined by matrix-assisted laser desorption MS to be Man3XylGlcNAc2. IgE binding to this protein was detected in sera of 41% of the allergic patients and 61% of the sensitized children. Partial N-terminal sequencing demonstrated similarity to legume storage proteins. The IgE reactivity of this structure was partially resistant to heating. The rat basophil leukaemia cell mediator release assay was used for estimation of cross-sensitization between hazelnut and birch pollen, confirming the finding that hazelnuts contain a heat-resistant allergenicity without cross-reactivity to birch pollen allergens
[41] - Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-570
Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd.
[42] - Wigotzki M, Steinhart H, Paschke A. Influence of Varieties, Storage and Heat Treatment on IgE-Binding Proteins in Hazelnuts (Corylus avellana). Food Agric Immunol 2000;12:217-229
The allergenicity of four hazelnut varieties (Runde Römer, Levantiner, Neapler, Contorta) and different types of heat treated and stored hazelnuts was examined and compared by SDSPAGE/immunoblotting and EAST-inhibition experiments using sera of 19 hazelnut allergic individuals. The immunoblot and EAST-inhibition investigations of the four varieties revealed only slight differences with regard to the allergenic activities. Heat treatment at 100°C for up to 90 min had no influence on the allergenicity of hazelnut prote ins. The IgE binding activity of the main hazelnut allergens decreased after 15 min heat treatment at temperatures between 100 and 185°C and was no longer detectable at 170°C. A protein < 14 kDa appeared to be very stable to heat and could be detected even after treatment at 185°C. Microwave treatment and storage of ground hazelnuts up to 19 weeks at room temperature had no influence on the allergenicity of hazelnut proteins.
[43] - Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-570
Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd.
[44] - Schocker F, Lüttkopf D, Müller U, Thomas P, Vieths S, Becker WM. IgE binding to unique hazelnut allergens: identification of non pollen-related and heat-stable hazelnut allergens eliciting severe allergic reactions. Eur J Nutr 2000;39:172-180
BACKGROUND: Usually hazelnut allergic patients suffer from the tree pollen associated oral allergy syndrome (OAS) caused by cross-reactive structures. Anaphylactic reactions elicited by hazelnuts happen rarely but are of high clinical significance. Considering that hazelnuts are ingredients in processed foods, hazelnuts may play an important role as hidden allergens for these high risk patients. Therefore, we analyzed the IgE reactivity of a young woman with severe allergic reactions after ingestion of hazelnuts without any association to tree pollen allergy. AIM OF THE STUDY: The aim of this study was to identify and characterize these potent hazelnut-specific allergens. We compared these allergens to structures displayed by sera from patients with a completely or partially non pollen-related hazelnut allergy and with birch pollen-related hazelnut allergy. None of the sera had a clinical history of anaphylaxis. Special emphasis was placed on the heat stability and cross-reactivity of these allergens. METHODS/RESULTS: Using Western blotting with extract from birch pollen and EAST inhibition techniques we were able to show that the allergens in the serum sample of the young woman were not cross-reactive with birch pollen. Immunoblot experiments with extracts from native and heated hazelnuts and EAST inhibition tests further characterized these allergens to be heat-stable. Unlike the IgE binding pattern of the sera from the patients with pollen-related hazelnut allergy, low molecular weight proteins below 10 kDa were identified by the sera from the patients without pollinosis. CONCLUSIONS: Since the binding pattern of the serum sample of the young woman was different from that of the sera from patients without pollen allergy but less severe symptoms, we assume an association between single non pollen-dependent hazelnut allergens in the low molecular range and severe allergic reactions. These results enable us to approach a subgroup of hazelnut allergens which we believe to be responsible for anaphylactic reactions in hazelnut allergic patients after ingestion of heat-stable hazelnut structures in processed food stuff, independent of pollinosis.
[45] - Wensing M, Penninks AH, Hefle SL, Akkerdaas JH, van Ree R, Koppelman SJ, et al. Determination of threshold levels of patients with hazelnut allergy using double-blind placebo-controlled food challenges (DBPCFC's). 8th International Symposium on Problems of Food Allergy, Venice 2001, March 11-13
Because hazelnuts are used in prepackaged foods, sometimes without clear notification on the label, accidental ingestion occurs. To evaluate the risks for hazelnut allergic patients after accidental ingestion of low quantities of hazelnut, it is important to reveal the distribution of threshold levels in hazelnut allergic patients. The aim of this study is to determine the distribution of threshold levels in hazelnut allergic patients using DBPCFC'S. Adult patients with hazelnut allergy, as determined by a positive history of adverse reactions, positive skin prick test (3 2 +) and/or elevated specific IgE-level (CAP3 class II) were included in the study. Challenges were performed using seven placebo doses and seven doses of roasted hazelnut, ranging from 30 µg to 30 mg of hazelnut protein, hidden in mashed potatoes. The schedule of seven placebo and verum doses was random with an interval of 30 minutes when no reactions occurred. A challenge was discontinued when objective reactions were observed or subjective reactions lasted for longer than one hour. The challenges were conducted in a clinical research setting equipped for monitoring of vital signs and resuscitation. Ethical approval for this study was obtained from the hospitals ethics committee. All patients gave written informed consent. The first DBPCFC'S of nine hazelnut allergic patients (4 women) were all negative. Since we were not sure whether the doses were too low or whether we should use raw instead of roasted hazelnuts, we investigated the heat lability of the 18 kD major hazelnut allergen, Cor a 1, that shows high homology to the major birch pollen allergen Bet v 1. Using a competitive RIA with monoclonal anti-Bet v 1, radiolabeled Bet v 1 and raw hazelnut meal (incubated with temperatures of 20 up to 100 °C) together with the roasted hazelnut meal, we demonstrated that binding of Cor a 1 was severely decreased after heating of 80 °C and higher. No activity of this allergen could be detected in the roasted hazelnut meal. Therefore, we decided to use raw hazelnut meal for the DBPCFC'S with slightly higher doses (1mg - 1g). The results of six rechallenged allergic patients are summarized in the table below. Reaction Threshold dose No 1mg 3mg 10mg 30 mg 100mg 300 mg 1g reaction Subjective (n) 1 1 0 2 2 0 0 0 Objective (n) 00000006 No objective reactions were observed. All subjective reactions consisted of itching of throat, tongue and/or lips and occurred within 2 to 10 minutes after ingestion of the test meal. Patients did not react to any placebo dose. Late reactions did not occur. From these preliminary results it appears that threshold levels for subjective reactions in hazelnut allergic patients vary from less than 1mg up to doses probably higher than 100mg of raw hazelnut protein. Another important result is the observed heat lability of the major hazelnut allergen, implicating that heating of hazelnuts decreases the allergenic potential for a substantial part of the hazelnut allergic patients.
[46] - Akkerdaas J, Schocker F, Alcocer M, Schilte P, Knulst A, Hefle S, et al. Detection and Immunological Characterization of Hazelnut 2S Albumin. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°1038
RATIONALE: The extremely stable storage protein 2S albumin has been identified as a potentially severe allergen in Brazil nut and walnut. As a ubiquitous component of plant seeds we aimed at identifying the expected homologous allergen from hazelnuts. METHODS: Crude, roasted and pepsin-digested hazelnut extracts were analyzed for the presence of 2S albumin by RAST(-inhibition) and immunoblot using human serum samples with known IgE reactivity to the Brazil nut 2S albumin (Ber e 1) and with rabbit anti-Ber e 1. Biological activity was assessed by histamine release tests (HRT). A cDNA library of hazelnut was constructed and screened with 2S albumin reactive serum samples. RESULTS: Immunoblot analysis with rabbit anti-Ber e 1 confirmed the presence of 2S albumin in hazelnut. This band was also recognized by serum IgE of hazelnut allergic patients with IgE reactivity to Ber e 1. Variable degrees of crossreactivity between both nut allergens was confirmed by RAST-inhibition. HRT showed biological activity of 2S albumin at concentration <1 ng/ml. Using serum of a patient with hazelnut anaphylaxis and IgE reactivity to Ber e 1, several putative hazelnut 2S albumin cDNA clones were picked up. CONCLUSIONS: 2S albumin is a heat and protease resistant allergen in hazelnut with variable degree of crossreactivity to Ber e 1. Expression of the putative clones will provide valuable tools for improvement of diagnostics for tree nut allergens.
[47] - Akkerdaas JH, Wensing M, van Leeuwen A, Schilte P, Hefle SL, Aalberse RC, et al. IgE Profiles in Dutch Hazelnut Allergic Patients: Non-pollen Related Recognition of Cor a 1 and Pepsin-induced Neo-epitopes. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°801
Rationale Dutch hazelnut allergic patients are typically sensitized to Cor a 1 and profilin on the basis of primary sensitization to birch pollen Bet v 1 and Bet v 2. These allergens are extremely pepsin-sensitive, thus limiting clinical allergy to OAS. The aim of this study was to investigate whether other (potentially stable) allergens play a role. Method s : Sera were selected from patients (n=60) with history- and/or DBPCFC-proven hazelnut allergy or from subjects (n=18) with high IgE titers to hazelnut but low or absent to apple and birch. Extracts of crude, roasted and pepsin-digested hazelnut were used to assess the stability of IgE epitopes. Extraction of hazelnut at pH2.5 resulted in enrichment for 2S albumin. These extracts and affinity-purified Cor a 1 were used in RAST and immunoblot analyses. Result s : As expected, in the group of patients with proven hazelnut allergy, IgE recognition of Cor a 1 dominated (58/60 with >0.3 IU/ml). In accordance with the lability of Cor a 1, recognition of roasted and pepsin-digested hazelnut was much less frequent: 36% and 12% positives, respectively. Two sera contained IgE antibodies that preferentially recognized hazelnut extract after pepsin-digestion, thus pointing towards neo-epitopes. Among the sera selected on low reactivity to birch/apple, 5/18 recognized hazelnut Cor a 1 but not pollen Cor a 1 or Bet v 1. Conclusions : Cor a 1 recognition independent from pollen sensitization exists, raising questions about its stability. Pepsin-digestion supports the existence of neo-epitopes.
[48] - Skamstrup Hansen K, Ballmer-Weber BK, Lüttkopf D, Skov PS, Wüthrich B, Bindslev-Jensen C, et al. Roasted hazelnuts – allergenic activity evaluated by double-blind, placebo-controlled food challenge. Allergy 2003;58:132-138
BACKGROUND: Allergy to hazelnuts is a common example of birch pollen related food allergy. Symptoms upon ingestion are often confined to the mouth and throat, but severe systemic reactions have been described in some patients. The aim of the study was to evaluate the reduction in allergenicity by roasting of the nuts . METHODS: Double-blind, placebo-controlled food challenges (DBPCFC) with roasted hazelnuts (140 degrees C, 40 min) were performed in 17 birch pollen allergic patients with DBPCFC-confirmed food allergy to raw hazelnuts. The effect of roasting was further evaluated by skin prick test (SPT), histamine release (HR), measurement of specific IgE, and IgE-inhibition experiments . RESULTS: In 5/17 patients the DBPCFC with the roasted nuts were positive. The symptoms were generally mild and included OAS (oral allergy syndrome) in all patients. Roasting of the nuts significantly reduced the allergenic activity evaluated by SPT, HR, specific IgE, and IgE-inhibition. Immunoblotting experiments with recombinant hazelnut allergens showed sensitization against Cor a 1.04 in 16/17 patients and against Cor a 2 in 7/17 patients. None of the patients were sensitized to Cor a 8. Challenge-positive patients did not differ from the rest in IgE-binding pattern . CONCLUSIONS: All the applied methods indicated that roasting of hazelnuts reduces the allergenicity, but since 5/17 birch pollen allergic patients were DBPCFC-positive to the roasted nuts, ingestion of roasted hazelnuts or products containing roasted hazelnuts can not be considered safe for a number of hazelnut allergic consumers. For patients with a history of severe allergic symptoms upon ingestion of hazelnuts, thorough and conscientious food labelling of hazelnuts and hazelnut residues is essential.
[49] - 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
[50] - Schimek EM, Zwölfer B, Briza P, Jahn-Schmid B, Vogel L, Vieths S, et al. Gastrointestinal digestion of Bet v 1-homologous food allergens destroys their mediator-releasing, but not T cell–activating, capacity. J Allergy Clin Immunol 2005;116:1327-1333
BACKGROUND: Food allergy to apples, hazelnuts, and celery is frequent in individuals with birch pollen allergy because IgE antibodies specific for the major birch pollen allergen, Bet v 1, cross-react with structurally related allergens in these foods. In addition, T lymphocytes specific for Bet v 1 also cross-react with these dietary proteins . OBJECTIVE: We sought to evaluate the effects of simulated gastrointestinal degradation of Bet v 1-related food allergens on their mediator-releasing and T cell-activating capacity . METHODS: Recombinant Mal d 1, Cor a 1.04, and Api g 1 were incubated separately with pepsin and trypsin. Binding of IgE was tested in immunoblots. After successive incubation with both enzymes, allergens were tested in mast cell mediator release assays and used to stimulate PBMCs and Bet v 1-specific T-cell lines and clones. Proteolytic fragments of allergens were analyzed and sequenced by means of mass spectrometry . RESULTS: Pepsin completely destroyed IgE binding of all allergens within 1 second, and trypsin completely destroyed IgE binding of all allergens within 15 minutes, except for the major hazelnut allergen, which remained intact for 2 hours of trypsinolysis. Allergens after gastrointestinal digestion did not induce basophil activation but induced proliferation in PBMCs from allergic and nonallergic individuals. Digested Mal d 1 and Cor a 1.04 still activated Bet v 1-specific T cells, whereas digested Api g 1 did not. Different proteolytic fragments of Mal d 1 and Cor a 1.04 matching relevant Bet v 1 T-cell epitopes were found . CONCLUSION: Gastrointestinal degradation of Bet v 1-related food allergens destroys their histamine-releasing, but not T cell-activating, property. Our data emphasize that birch pollen-related foods are relevant activators of pollen-specific T cells.
[51] - Untersmayr E, Poulsen L, Vestergaard H, Malling H, Jensen L, Platzer M, et al. Gastric hypoacidity lowers threshold levels for food allergens in vitro and in vivo. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1509
Background: We could show previously that gastric acid-suppression with antacids or anti-ulcer drugs promotes the development of type I food allergy. In the present study we aimed to investigate the role of gastric digestion in already established food allergy. Methods and Results: The reactivity pattern and biological activity of digested and undigested codfish proteins were analysed in SDS-PAGE, IgE immunoblot, RAST inhibition experiments and histamine release assays with sera of codfish allergic patients. These in vitro testing revealed that physiologically degraded codfish proteins exhibit an up to 10.000-fold decreased IgE-binding capacity and trigger histamine release only at relatively high concentrations. Furthermore, codfish allergic patients were enrolled in skin prick tests and double-blind placebo-controlled food challenges with codfish samples subjected in vitro digestion at pH 2.0 and 3.0. The codfish samples incubated with gastric enzymes at low pH induced a significantly reduced wheal reaction, whereas treatment at elevated pH hindered digestion and rendered a similar reaction as even undigested extracts. In the blinded food challenges the allergic patients revealed a 10- to 30-fold lower tolerated cumulative fish protein dose when the samples were subjected pepsin incubation at elevated pH. Conclusion: Our data strongly suggest that situations of insufficient gastric digestion, as occurring under acid-suppression therapy, represent a risk for allergic patients as threshold levels of food allergens for triggering anaphylaxis are likely to be significantly lowered.
[52] - Schöll I, Untersmayr E, Bakos N, Roth-Walter F, Gleiss A, Boltz-Nitulescu G, et al. Antiulcer drugs promote oral sensitization and hypersensitivity to hazelnut allergens in BALB/c mice and humans. Am J Clin Nutr 2005;81:154-160
BACKGROUND: Hazelnut allergy can be a consequence of sensitization to cross-reactive pollen, especially from the Fagales family. However, severe allergic reactions after ingestion of hazelnuts without associated pollen allergy have been reported. In these cases, oral sensitization by hazelnut ingestion is plausible. OBJECTIVE: We have reported that antiulcer drugs promote oral sensitization to digestion-labile food allergens. Because hazelnut proteins were sensitive to gastric digestion in our in vitro assay, we aimed to analyze the effect of antiulcer treatment on oral sensitization to hazelnut proteins. DESIGN: BALB/c mice were fed hazelnut extract with or without antiulcer drugs. In parallel, gastroenterologic patients (n = 153) were screened during antiulcer treatment for specific immunoglobulin (Ig) E to hazelnut and inhalative allergens in vitro and in vivo. RESULTS: Mice fed hazelnut extract in combination with antiulcer drugs formed anaphylactogenic IgG1 toward hazelnut and developed type I skin reactivity to hazelnut extract. In the human study population, 5 of 153 (3.3%) patients developed hazelnut-specific IgE, 4 of 5 developed specific skin reactivity, 3 of 5 had a positive result to oral provocation, and 2 of 5 manifested a food allergy to hazelnut after a 3-mo course of antiulcer treatment. Immunoblot testing with recombinant allergens showed that hazelnut, but not Fagales pollen, was the genuine elicitor in mice and humans. CONCLUSION: Our experimental and epidemiologic data suggest that the intake of antiulcer drugs may lead to the induction of immediate-type food hypersensitivity toward hazelnut.
[53] - Schöll I, Untersmayr E, Bakos N, Roth-Walter F, Gleiss A, Boltz-Nitulescu G, et al. Antiulcer drugs promote oral sensitization and hypersensitivity to hazelnut allergens in BALB/c mice and humans. Am J Clin Nutr 2005;81:154-160
BACKGROUND: Hazelnut allergy can be a consequence of sensitization to cross-reactive pollen, especially from the Fagales family. However, severe allergic reactions after ingestion of hazelnuts without associated pollen allergy have been reported. In these cases, oral sensitization by hazelnut ingestion is plausible. OBJECTIVE: We have reported that antiulcer drugs promote oral sensitization to digestion-labile food allergens. Because hazelnut proteins were sensitive to gastric digestion in our in vitro assay, we aimed to analyze the effect of antiulcer treatment on oral sensitization to hazelnut proteins. DESIGN: BALB/c mice were fed hazelnut extract with or without antiulcer drugs. In parallel, gastroenterologic patients (n = 153) were screened during antiulcer treatment for specific immunoglobulin (Ig) E to hazelnut and inhalative allergens in vitro and in vivo. RESULTS: Mice fed hazelnut extract in combination with antiulcer drugs formed anaphylactogenic IgG1 toward hazelnut and developed type I skin reactivity to hazelnut extract. In the human study population, 5 of 153 (3.3%) patients developed hazelnut-specific IgE, 4 of 5 developed specific skin reactivity, 3 of 5 had a positive result to oral provocation, and 2 of 5 manifested a food allergy to hazelnut after a 3-mo course of antiulcer treatment. Immunoblot testing with recombinant allergens showed that hazelnut, but not Fagales pollen, was the genuine elicitor in mice and humans. CONCLUSION: Our experimental and epidemiologic data suggest that the intake of antiulcer drugs may lead to the induction of immediate-type food hypersensitivity toward hazelnut.
[54] - Akkerdaas JH, Wensing M, Asero R, Fernandez Rivas M, Knulst AC, Bolhaar S, et al. IgE Binding to Pepsin-Digested Food Extracts. Int Arch Allergy Immunol 2005;138:203-208
BACKGROUND: Pepsin resistance of allergens like lipid transfer protein and 2S albumin has been suggested as explanation for the severity of symptoms often induced by these allergens. Component-resolved diagnosis with purified labile and stable allergens has therefore been proposed to better characterize the risk involved in a positive in vitro IgE test. However, for many foods, purified allergens are not (yet) available. OBJECTIVE: It was the aim of this study to evaluate the potential of pepsin-digested whole-food extracts to distinguish between IgE responses to stable (potentially severe) and labile (mild) allergens. METHODS: Sera (n = 143) from Italian, Spanish and Dutch patients with hazelnut and/or apple ingestion-related symptoms were analyzed for residual IgE binding to pepsin-resistant hazelnut and/or apple allergens. Control and pepsin-digested hazelnut and apple extracts were used for radioallergosorbent test analysis and immunoblot analysis. RESULTS: Pepsin digestion of food extracts, like from hazelnut and apple used for in vitro diagnostic tests, provides a way to distinguish sensitization to pepsin-resistant allergens from that to pepsin-susceptible allergens. In this selected group of patients, IgE reactivity to pepsin-digested extracts correlated with sensitization to the stable allergen lipid transfer protein. The analysis further revealed that the use of soluble pepsin can result in false-positive in vitro tests (2/143). CONCLUSION: Pepsin-digested food extracts are a convenient tool to identify patients with IgE antibodies against potentially dangerous stable allergens, in particular for those foods where the relevant stable allergens have not yet been identified. This can increase the clinical prognostic value of food allergy serology.
[55] - Untersmayr E, Bakos N, Schöll I, Kundi M, Roth-Walter F, Szalai K, et al. Anti-ulcer drugs promote IgE formation toward dietary antigens in adult patients. FASEB J 2005;19:656-658
Recently, we have demonstrated that anti-ulcer drugs, such as H2-receptor blockers and proton pump inhibitors, promote the development of immediate type food allergy toward digestion-labile proteins in mice. The aim of this study was to examine the allergological relevance of these findings in humans. In an observational cohort study, we screened 152 adult patients from a gastroenterological outpatient clinic with negative case histories for atopy or allergy, who were medicated with H2-receptor blockers or proton pump inhibitors for 3 months. IgE reactivities to food allergens before and after 3 months of anti-acid treatment were compared serologically. Ten percent of the patients showed a boost of preexisting IgE antibodies and 15% de novo IgE formation toward numerous digestion-labile dietary compounds, like milk, potato, celery, carrots, apple, orange, wheat, and rye flour. Thus, the relative risk to develop food-specific IgE after anti-acid therapy was 10.5 (95% confidence interval: 1.44-76.48). The long-term effect was evaluated 5 months after therapy. Food-specific IgE could still be measured in 6% of the patients, as well as significantly elevated serum concentrations of ST2, a Th2-specific marker. An unspecific boost during the pollen season could be excluded, as 50 untreated control patients revealed no changes in their IgE pattern. In line with our previous animal experiments, our data strongly suggest that anti-ulcer treatment primes the development of IgE toward dietary compounds in long-term acid-suppressed patients.
[56] - Benito C, González-Mancebo E, de Durana MD, Tolón RM, Fernández-Rivas M. Identification of a 7S globulin as a novel coconut allergen. Ann Allergy Asthma Immunol 2007;98:580-584
BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.
[57] - Pallares DE. Allergy to macadamia nut. Ann Allergy Asthma Immunol 2000;85:385-386
Nuts are one of the most common foods causing allergic reactions in children and adults although an allergic reaction to macadamia nut has been reported only once previously. OBJECTIVE: To report a case of macadamia nut-induced allergic reaction. METHODS: Skin prick test with macadamia nut was performed in a private practice office. RESULTS: The results demonstrated type-I hypersensitivity reaction with macadamia nut. CONCLUSION: Macadamia nut ingestion can cause immediate-type allergic reactions.
[59] - Malanin K, Lundberg M, Johansson SG. Anaphylactic reaction caused by neoallergens in heated pecan nut. Allergy 1995;50:988-991
An atopic girl experienced an anaphylactic reaction after eating cookies containing pecan nuts. Investigations revealed that she had developed IgE antibodies exclusively specific against allergenic determinants present in aged or heated pecan nuts, but not in fresh pecans. These neoallergenic determinants were located on protein(s) with a molecular weight of approximately 15 kDa. Neoallergens appearing during heating or storing of foods may be important in some anaphylactic reactions.
[61] - Venkatachalam M, Teuber SS, Peterson WR, Roux KH, Sathe SK. Antigenic Stability of Pecan [Carya illinoinensis (Wangenh.) K. Koch] Proteins: Effects of Thermal Treatments and in Vitro Digestion. J Agric Food Chem 2006;54:1449-1458
Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.
[62] - Malanin K, Lundberg M, Johansson SG. Anaphylactic reaction caused by neoallergens in heated pecan nut. Allergy 1995;50:988-991
An atopic girl experienced an anaphylactic reaction after eating cookies containing pecan nuts. Investigations revealed that she had developed IgE antibodies exclusively specific against allergenic determinants present in aged or heated pecan nuts, but not in fresh pecans. These neoallergenic determinants were located on protein(s) with a molecular weight of approximately 15 kDa. Neoallergens appearing during heating or storing of foods may be important in some anaphylactic reactions.
[63] - Venkatachalam M, Teuber SS, Peterson WR, Roux KH, Sathe SK. Antigenic Stability of Pecan [Carya illinoinensis (Wangenh.) K. Koch] Proteins: Effects of Thermal Treatments and in Vitro Digestion. J Agric Food Chem 2006;54:1449-1458
Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.
[64] - Murtagh GJ, Dumoulin M, Archer DB, Alcocer MJ. Stability of recombinant 2 S albumin allergens in vitro. Biochem Soc Trans 2002;30:913-915
Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response
[65] - Visitsunthorn N, Tiranathanakul A, Netrakul R, Vichyanond P. Evaluation of consistency between local and imported seafood allergen extracts. J Med Assoc Thai 2002;85(Suppl. 2):S593-S598
BACKGROUND: Seafood is a common cause of food allergy in Thai adults and children. Skin prick test is a safe and convenient way to screen seafood allergy. To date, the Allergy Unit, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University uses imported extracts for seafood skin prick test. The extracts are expensive and may not be the same species as seafood in Thailand. OBJECTIVES: To compare the consistency between local seafood allergen extracts prepared by the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University and imported seafood allergen extracts. METHODS: The study was a prospective comparative trial performed in children who attended the Pediatric Allergy Clinic, Siriraj Hospital from March 1999 to October 2000. The skin prick test was performed with the local seafood allergen extracts prepared by a pharmacist from the Department of Pharmacology and the imported seafood allergen extracts included shrimp, fish and crab. Histamine and normal saline were used as positive and negative control respectively. The positive result was recorded when wheal reaction was > or = 3 mm larger than negative control. RESULTS: Eighty eight patients (57 boys and 31 girls) were included in this study. The average age was 7.7 years (1-15 years). Half of the patients had a history of seafood allergy. The study showed probable consistency between imported and local skin prick test of shrimp and crab extracts (kappa = 5-7) but no consistency between imported and local skin prick test of fish extracts (kappa < 5). The study also showed no consistency between history of seafood and skin prick test result. CONCLUSION: Local seafood allergen extracts from the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University are safe but they cannot replace the imported seafood allergen extracts. Further investigations about sensitivity and specificity of both kinds of allergen extracts are necessary.
[66] - Koppelman SJ, Nieuwenhuizen WF, Gaspari M, Knippels LM, Penninks AH, Knol EF, et al. Reversible denaturation of Brazil nut 2S albumin (Ber e1) and implication of structural destabilization on digestion by pepsin. J Agric Food Chem 2005;53:123-131
The high resistance of Brazil nut 2S albumin, previously identified as an allergen, against proteolysis by pepsin was examined in this work. Although the denaturation temperature of this protein exceeds the 110 degrees C at neutral pH, at low pH a fully reversible thermal denaturation was observed at approximately 82 degrees C. The poor digestibility of the protein by pepsin illustrates the tight globular packing. Chemical processing (i.e., subsequent reduction and alkylation of the protein) was used to destabilize the globular fold. Far-UV circular dichroism and infrared spectroscopy showed that the reduced and alkylated form had lost its beta-structures, whereas the alpha-helix content was conserved. The free energy of stabilization of the globular fold of the processed protein as assessed by a guanidine titration study was only 30-40% of that of the native form. Size exclusion chromatography indicated that the heavy chain lost its globular character once separated from the native 2S albumin. The consequences of these changes in structural stability for degradation by pepsin were analyzed using gel electrophoresis and mass spectrometry. Whereas native 2S albumin was digested slowly in 1 h, the reduced and alkylated protein was digested completely within 30 s. These results are discussed in view of the potential allergenicity of Brazil nut 2S albumin.
[67] - Koppelman SJ, Nieuwenhuizen WF, Gaspari M, Knippels LMJ, Penninks AH, Knol EF, et al. Stability of protein structure protects Brazil nut 2S albumin (Ber e1) from digestion by pepsin. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1016
2S albumin from Brazil nut was purified using selective extraction and cation-exchange chromatography. This purified protein could be reduced and alkylated and the heavy and light chain were separated using size-exclusion chromatography. The secondary structure of native and reduced and alkylated 2S albumin was investigated with far UV Circular dichroism and infrared spectroscopy. Compared to native 2S albumin, the reduced and alkylated form had lost its b-structures, whereas the a- helix content was maintained. The free energy of stabilization of the globular structure of the reduced and alkylated form as assessed by a guadinium titration study was only 30-40% of that of the native form. Heat-induced conformational changes of the native protein occurred at 82 °C, and were fully reversible. Additional information on the tertiary and quaternary structure was obtained from size-exclusion chromatography, and indicated that the heavy chain lost its globular character once separated from the native 2S albumin. The consequences of these structural changes for the digestion by pepsin were analysed. SDS-PAGE showed that native 2S albumin degraded gradually on a time scale of an hour, whereas the reduced and alkylated form disappeared within 30 seconds under the same experimental conditions with the heavy chain being more susceptible to proteolysis compared to the light chain. We conclude that the secondary structure and tertiary/quaternary structure of 2S albumin is stabilized by disulfide bonds, and that destabilization of the structure leads to a pronounced pepsin susceptibility. These results are discussed in view of the potential allergenicity of Brazil nut 2S albumin.
[68] - Burnett GR, Wickham M, Fillery-Travis A, Robertson JA, Belton PS, Gilbert SM, et al. Interaction between protein allergens and model gastric emulsions. Biochem Soc Trans 2002;30:916-918
The observed resistance to pepsinolysis of known food allergens has been suggested as a predictor of their allergenic risk. Consequently, resistance to pepsinolysis has become incorporated into decision tree assessment for potential allergenic risk posed by novel foods. However, existing methods take little account of the interaction between food structure and physiological conditions existing during digestion in vivo. Here we show that a range of protein allergens can adsorb to model stomach emulsions, providing a further means of resisting digestion. We also show that raising the pH and the addition of bile salts to a model stomach emulsion, thereby mimicking the duodenal environment, has the effect of desorbing the adsorbed protein.
[69] - Alcocer MJC, Murtagh GJ, Bailey K, Dumoulin M, Sarabia Meseguer A, Parker MJ, et al. The Disulphide Mapping, Folding and Characterisation of Recombinant Ber e 1, an Allergenic Protein, and SFA8, Two Sulphur-rich 2S Plant Albumins. J Mol Biol 2002;324:165-175
We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.
[70] - Moreno FJ, Mellon FA, Wickham MS, Bottrill AR, Mills EN. Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion. FEBS J 2005;272:341-352
The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.
[71] - Bock SA, Atkins FM. The natural history of peanut allergy. J Allergy Clin Immunol 1989;83:900-904
Between 1973 and 1985, 114 children, aged 2 to 14 years, underwent double-blind, placebo-controlled, food challenge (DBPCFC) to peanut. Thirty-two of 46 children with symptoms produced by DBPCFC to peanut were included in this longitudinal evaluation. Contact was made with the 32 subjects 2 to 14 years after their positive DBPCFC to peanut. All 32 subjects had exhibited a positive puncture skin test to peanut at the time of the original evaluation. Sixteen subjects had experienced symptoms caused by accidental peanut ingestion in the year before contact. Eight subjects had reacted to accidental ingestion in more than 1 year but less than 5 years before contact. Eight subjects had completely avoided peanut since the original evaluation and positive DBPCFC. No subjects could be demonstrated to have "outgrown" their peanut reactivity. All subjects tested continued to have skin reactivity to a puncture skin test with peanut extract. It appears uncommon for peanut-sensitive patients to lose their clinical reactivity, even after many years have elapsed. In addition, data were collected concerning reactions to other legumes and other (nonlegume) nuts. Only two patients with DBPCFC to peanut reacted on DBPCFC to soy or pea (one each). None of the subjects with a positive DBPCFC to peanut reacted to nonlegume nuts.
[72] - Rancé F, Bidat E, Bourrier T, Sabouraud D. Cashew allergy: observations of 42 children without associated peanut allergy. Allergy 2003;58:1311-1314
BACKGROUND: Cashew allergy seems to be increasingly frequent. The goal of the present study was to analyse the clinical features and results of investigations of 42 children with cashew allergy . METHODS: The clinical features and results of skin prick tests, specific IgE assays, and food challenges were analysed . RESULTS: The mean age at first allergic reaction was 2 years and the mean age at diagnosis of cashew allergy was 2.7 years. One in five children (12%) had a prior history of exposure to cashew nuts. Fifty-six per cent had skin symptoms, 25% had respiratory signs and 17% had digestive signs. Eighteen children had proven, associated food allergies (pistachio, seven; egg, five; mustard, three; shrimp, two; cow milk, one). The mean wheal diameter of the skin prick tests was 7 mm (3-16 mm) and the mean specific IgE level was 3.1 kUA/L (<0.35->100 kUA/L). Eight children had positive food challenges . CONCLUSION: The increase in cashew allergy is worrying because it affects young children who may have a reaction without ever having been exposed to cashews. Almost one-third of children are allergic to pistachios, which belong to the same botanical family as cashews. Clinical history is generally and sufficiently suggestive to diagnose cashew allergy without recourse to food challenges.
[73] - Hourihane JO, Harris H, Langton-Hewer S, Kilburn SA, Warner JO. Clinical features of cashew allergy. Allergy 2001;56:252-253
This study reports on the findings of cashew allergy in 29 patients. 48% reacted to minimal contact with cashew, i.e., smelling, touching, or tasting, but not eating cashew. The authors point out that severe reactions are as severe as those from peanut allergy.
[76] - Fleischer DM, Conover-Walker MK, Matsui EC, Wood RA. The natural history of tree nut allergy. J Allergy Clin Immunol 2005;116:1087-1093
BACKGROUND: Although 20% of children outgrow peanut allergy, the natural history of tree nut (TN) allergy has not been well studied . OBJECTIVE: The goals of the study were to estimate the proportion of children who outgrow TN allergy and examine predictors of outgrowing it . METHODS: Patients with TN allergy, defined as a history of reaction on ingestion and evidence of TN-specific IgE (TN-IgE) or positive TN-specific IgE level but no history of ingestion, were evaluated. If all current TN-IgE levels were less than 10 kilounits of antibody (kU(A))/L, double-blind, placebo-controlled food challenges were offered. Patients who had undergone open TN challenges as part of routine clinical care were also included . RESULTS: Two hundred seventy-eight patients with TN allergy were identified. One hundred one (36%) had a history of acute reactions, 12 (12%) of whom had reactions to multiple TNs and 73 (63%) of whom had a history of moderate-to-severe reactions. Nine of 20 patients who had previously reacted to a TN passed challenges, so that 9 (8.9%; 95% CI, 4% to 16%) of 101 patients with a history of prior TN reactions outgrew TN allergy. Fourteen of 19 who had never ingested TNs but had detectable TN-specific IgE levels passed challenges. One hundred sixty-one did not meet the challenge criteria, and 78 met the criteria but declined challenges. Looking at specific TN-IgE cutoffs, 58% with TN-IgE levels of 5 kU(A)/L or less and 63% with TN-IgE levels of 2 kU(A)/L or less passed challenges . CONCLUSIONS: Approximately 9% of patients outgrow TN allergy, including some who had prior severe reactions. Although ideal cutoffs for challenge cannot be firmly recommended on the basis of these data, patients aged 4 years or older with all TN-IgE levels of 5 kU(A)/L or less should be considered for challenge.
[77] - Clark AT, Ewan PW. The development and progression of allergy to multiple nuts at different ages. Pediatr Allergy Immunol 2005;16:507-511
The aims of this study were to define the development of sensitization and clinical allergy to multiple nut types by age, and to identify associations. This observational cross- sectional study was carried out in a tertiary level allergy clinic. Seven hundred and eighty four nut-allergic children, prospectively enrolled on first attendance with history of a type-1 allergic reaction shortly after definite nut ingestion with evidence of sensitization (presence of nut-specific IgE) by skin prick test (SPT) to peanut, Brazil, almond, hazel and walnut (positive > or = 3 mm). Details of exposure (tolerance or allergy) to each nut were obtained. The main outcome measures were mono or multi-sensitization (specific IgE to one or more than one nut type, demonstrated by SPT); mono or multi-allergy (clinical allergy to one or more than one nut type). By 2 yr of age at least 19% were multi-sensitized, and 2% multi-allergic. Increasing proportions were exposed to multiple nut types with increasing age (23% at 2 yr to 73% by 10 yr) and greater proportions were multi-sensitized (19% at 2 yr to 86% at 5-14 yr) and multi-allergic (2% at 2 yr to 47% at 14 yr). This study is the first to define the natural history of multiple nut allergies in childhood. New findings are that a large proportion of those aged 0-1 yr with nut allergy are already sensitized (have specific IgE) to multiple nut types, implying in utero or early life sensitization; those who present later in childhood are increasingly likely to be sensitized and clinically allergic to multiple nuts. This is related to increased duration of allergy and exposure to multiple nut types with age. Children with nut allergy should avoid all nut types from the onset.
[78] - Robotham JM, Wang F, Seamon V, Teuber SS, Sathe SK, Sampson HA, et al. Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family. J Allergy Clin Immunol 2005;115:1284-1290
BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins . OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin . METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes . RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology . CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.
[80] - Skamstrup Hansen K, Ballmer-Weber BK, Lüttkopf D, Skov PS, Wüthrich B, Bindslev-Jensen C, et al. Roasted hazelnuts – allergenic activity evaluated by double-blind, placebo-controlled food challenge. Allergy 2003;58:132-138
BACKGROUND: Allergy to hazelnuts is a common example of birch pollen related food allergy. Symptoms upon ingestion are often confined to the mouth and throat, but severe systemic reactions have been described in some patients. The aim of the study was to evaluate the reduction in allergenicity by roasting of the nuts . METHODS: Double-blind, placebo-controlled food challenges (DBPCFC) with roasted hazelnuts (140 degrees C, 40 min) were performed in 17 birch pollen allergic patients with DBPCFC-confirmed food allergy to raw hazelnuts. The effect of roasting was further evaluated by skin prick test (SPT), histamine release (HR), measurement of specific IgE, and IgE-inhibition experiments . RESULTS: In 5/17 patients the DBPCFC with the roasted nuts were positive. The symptoms were generally mild and included OAS (oral allergy syndrome) in all patients. Roasting of the nuts significantly reduced the allergenic activity evaluated by SPT, HR, specific IgE, and IgE-inhibition. Immunoblotting experiments with recombinant hazelnut allergens showed sensitization against Cor a 1.04 in 16/17 patients and against Cor a 2 in 7/17 patients. None of the patients were sensitized to Cor a 8. Challenge-positive patients did not differ from the rest in IgE-binding pattern . CONCLUSIONS: All the applied methods indicated that roasting of hazelnuts reduces the allergenicity, but since 5/17 birch pollen allergic patients were DBPCFC-positive to the roasted nuts, ingestion of roasted hazelnuts or products containing roasted hazelnuts can not be considered safe for a number of hazelnut allergic consumers. For patients with a history of severe allergic symptoms upon ingestion of hazelnuts, thorough and conscientious food labelling of hazelnuts and hazelnut residues is essential.
[81] - van Odijk J, Bengtsson U. The fat content may affect the reaction pattern in double blind placebo controlled food challenges for hazelnut. Allergy 2007;62(suppl. 83):105
Background: Few studies have been performed regarding how nutrient composition, for example the fat content, may affect the outcome of the allergic reaction. To test the allergenicity of products with different fat contents, patients with a lower risk of severe reactions, for example birch pollen allergic patients with oral allergy syndrome to hazelnuts, may be involved. The aim was to study if the fat content in blinded food challenges to hazelnut affects the reaction pattern in birch pollen allergic patients with reported oral allergy syndrome to hazelnut. A secondary aim was to develop 2 recipes with different fat content that was documented blind by panels using a triangle test. METHODS: Two recipes containing the same amount of hazelnut but different content of fat were developed and tested on a panel of 24 subjects with triangle tests. The fat recipe contained 28.5 % of fat and the low-fat recipe 8.5 % of fat. The recipes were tested on separate blinded food challenges on 15 patients with documented birch pollen allergy and a history of oral allergy syndrome to hazelnuts. Onset and severity of symptoms were tested using a stopwatch and a VAS-scale. RESULTS: Both recipes were regarded equal regarding taste and texture as tested by the taste panels. All 15 subjects reacted to hazelnut challenge within 7.5 minutes as expected with itching in the oral cavity and swollen feeling of tongue and lips (oral allergy syndrome), regardless of fat content. All reactions terminated within 25 minutes. Twelve out of 15 subjects reacted with a shorter onset of symptoms when challenged with the low-fat recipe (p< 0.03). For 2 subjects, the onset was shorter for the fat recipe, and one subjects reacted within the same time range for both recipes (difference less than 5 seconds). There was a wider range of reactions reported by VAS scale to the fat recipe compared to non-fat recipe. There was no correlation between severity of symptoms (measured by VAS scale) and specific IgE level to birch or hazelnut. There seems to be a delayed reaction pattern to high fat hazelnut challenges in birch pollen allergic patients. This result is in line with earlier studies on peanut challenges and may reflect the role of food composition in allergic reactions.
[82] - Akkerdaas JH, Wensing M, Knulst AC, Krebitz M, Breiteneder H, de Vries S, et al. How Accurate and Safe Is the Diagnosis of Hazelnut Allergy by Means of Commercial Skin Prick Test Reagents ? Int Arch Allergy Immunol 2003;132:132-140
BACKGROUND: Allergy to tree nuts, like hazelnuts, ranks among the most frequently observed food allergies. These allergies can start at early childhood and are, in contrast to other food allergies, not always outgrown by the patient. Tree nut allergy is frequently associated with severe reactions. Diagnosis partially relies on in vivo testing by means of a skin prick test (SPT) using commercially available SPT reagents . METHODS: Protein and allergen composition of nine commercial SPT solutions was evaluated using standard protein detection methods and specific immunoassays for measurement of five individual allergens. Diagnostic performance was assessed by SPT in 30 hazelnut-allergic subjects, of which 15 were provocation proven . RESULTS: Protein concentrations ranged from 0.2-14 mg/ml. SDS-PAGE/silver staining revealed clear differences in protein composition. The major allergen Cor a 1 was present in all extracts but concentrations differed up to a factor 50. An allergen associated with severe symptoms, Cor a 8 (lipid transfer protein), was not detected on immunoblot in three products, and concentrations varied by more than a factor 100 as was shown by RAST inhibition. Similar observations were made for profilin, thaumatin-like protein and a not fully characterized 38-kD allergen. Ratios of individual allergens were variable among the nine extracts. SPT showed significant difference, and 6/30 patients displayed false-negative results using 3/9 products . CONCLUSION: Variability in the composition of products for the diagnosis of hazelnut allergy is extreme. Sometimes, allergens implicated in severe anaphylaxis are not detected by immunoblotting. These shortcomings in standardisation and quality control can potentially cause a false-negative diagnosis in subjects at risk of severe reactions to hazelnuts.
[83] - Akkerdaas JH, Wensing M, Asero R, Fernandez Rivas M, Knulst AC, Bolhaar S, et al. IgE Binding to Pepsin-Digested Food Extracts. Int Arch Allergy Immunol 2005;138:203-208
BACKGROUND: Pepsin resistance of allergens like lipid transfer protein and 2S albumin has been suggested as explanation for the severity of symptoms often induced by these allergens. Component-resolved diagnosis with purified labile and stable allergens has therefore been proposed to better characterize the risk involved in a positive in vitro IgE test. However, for many foods, purified allergens are not (yet) available. OBJECTIVE: It was the aim of this study to evaluate the potential of pepsin-digested whole-food extracts to distinguish between IgE responses to stable (potentially severe) and labile (mild) allergens. METHODS: Sera (n = 143) from Italian, Spanish and Dutch patients with hazelnut and/or apple ingestion-related symptoms were analyzed for residual IgE binding to pepsin-resistant hazelnut and/or apple allergens. Control and pepsin-digested hazelnut and apple extracts were used for radioallergosorbent test analysis and immunoblot analysis. RESULTS: Pepsin digestion of food extracts, like from hazelnut and apple used for in vitro diagnostic tests, provides a way to distinguish sensitization to pepsin-resistant allergens from that to pepsin-susceptible allergens. In this selected group of patients, IgE reactivity to pepsin-digested extracts correlated with sensitization to the stable allergen lipid transfer protein. The analysis further revealed that the use of soluble pepsin can result in false-positive in vitro tests (2/143). CONCLUSION: Pepsin-digested food extracts are a convenient tool to identify patients with IgE antibodies against potentially dangerous stable allergens, in particular for those foods where the relevant stable allergens have not yet been identified. This can increase the clinical prognostic value of food allergy serology.
[84] - Andersson K, Vieths S, Lüttkopf D, Östling J, Lidholm J. Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking. Allergy Clin Immunol Int 2005;17(Suppl. 1):361
Background: Allergy to hazelnut belongs to the most common food allergies. Symptoms upon ingestion are typically local but may in some cases be systemic and even life-threatning. Accurate assessment of hazelnut sensitization is a vital part of patient care and dietary risk elimination. Several hazelnut proteins have been identified as allergens and characterised. A major allergen in central and northern European populations is Cor a 1.04, a member of the PR-10 protein family, while Cor a 8, a lipid transfer protein, appears particularly important in the south of Europe. Questions have been raised as to whether natural hazelnut extract contains sufficient amounts of all relevant allergen components to allow detection of hazelnut sensitization with fully adequate sensitivity. Methods: An assessment of allergen content in hazelnut extract was performed using selected patients' sera and allergen-specific rabbit antisera. Drawing from those results, experiments were performed to examine whether spiking of hazelnut extract with recombinant allergen could generate an allergen reagent with enhanced capacity to detect and quantify specific IgE antibodies. All IgE measurements were performed using the ImmunoCAP system. Result: Serial dilution experiments indicated Cor a 1 to be the least abundant in hazelnut extract of the allergen components studied. Spiking of hazelnut extract with recombinant Cor a 1.04 prior to solid phase coupling gave rise to significantly enhanced IgE antibody binding from Cor a 1.04 reactive sera. Dilution of the natural hazelnut extract prior to spiking, to reduce competition for solid phase attachment by abundant native components, further increased IgE binding. Neither of these extract manipulations were found to negatively affect IgE measurement in hazelnut reactive sera that were Cor a 1.04 negative, presumably due to the high-capacity solid phase of the assay used. Conclusion: A hazelnut allergen reagent with considerably enhanced capacity for specific IgE detection can be generated by supplementing natural extract with recombinant Cor a 1.04.
[85] - Büyüköztürk S, Kardes BA, Gelincik AA, Dal M, çolakoglu B, Akkor A, et al. Does Working in a Hazelnut Processing Factory Increase the Risk of Hazel Pollen and Nut Sensitivity ? Allergol Int 2005;54:469-472
This study showed that occupational exposure to hazelnut caused skin sensitivity to hazel and birch pollen, but this was not associated with an enhanced risk for allergic diseases.
[86] - Akkerdaas JH, Schocker F, Vieths S, Versteeg S, Zuidmeer L, Hefle SL, et al. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library. Mol Nutr Food Res 2006;50:18-23
The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.
[87] - Akkerdaas J, Wensing M, Knulst A, Aalberse R, Hefle S, van Ree R. Identification and characterisation of hazelnut allergens as LTP and 2S albumin. 8th International Symposium on Problems of Food Allergy, Venice 2001, March 11-13
Introduction: Hazelnut hypersensitivity is often observed in birch pollinosis. The major sensitising allergen is Bet v 1, which crossreacts with its homologue found in hazelnut, Cor a 1. Cor a 1 almost exclusively causes mild symptoms of the oral cavity (OAS). It is generally believed that only more stable allergens can reach the gastro-intestinal tract in an allergenic form, thereby having the potential to cause more severe symptoms. The aim of this study was to identify these (non-pollen related) stable allergens and compare their stability to Cor a 1. Materials and methods: Hazelnut extracts were prepared in two ways: 1) PVPP-method à la Björksten 2) at pH 2.51. Stability was studied by treatment with pepsin2. Extracts were analysed by SDS-PAGE/silverstaining. Immune-reactivity was studied by RAST, RAST-inhibition, immunoblotting and competitive RIA, using serum samples of hazelnut allergic patients, monoclonals and monospecific rabbit antisera. Results: Extraction of hazelnut at pH2,5 resulted in almost exclusively LMW proteins (28 kDa and around 10 kDa) in contrast to the conventional PVPP-extraction in which these bands were underrepresented among the broad range of protein bands present. The pH2.5-extracted proteins were shown to contain LTP by immunoblot with a crossreactive rabbit antiserum raised with carrot LTP. In contrast, Cor a 1 was almost absent as was shown by competitive RIA with Cor a 1 reactive mAb and by immunoblotting with human IgE. LTP showed unchanged immune-reactivity after pepsin digestion with human IgE. The same treatment resulted in almost complete loss of Cor a 1 reactivity. A rabbit antiserum against 2S albumin of Brasil nut3 (Ber e 1) showed reactivity to a 10 kDa band in both PVPP and pH2.5 extract. After pepsin digestion 2S albumin was still detected. Some hazelnut allergic sera were shown to contain Ber e 13 reactive IgE. RAST-inhibition revealed that a crossreactive 2S albumin was also recognised in hazelnut extract. Clinical data suggest that there is a trend for more severe symptoms in patients recognising stable allergens extractable at low pH. This needs further confirmation. Conclusions: Hazelnut extract contains both labile (Cor a 1) and stable allergens. Extractability of these allergens clearly differs. Two of the stable allergens in hazelnut were identified as LTP and 2S albumin. Stability appears to be an important parameter for distinction between mild (OAS) and potentially severe symptomatology.
[88] - van Odijk J, Bengtsson U. The fat content may affect the reaction pattern in double blind placebo controlled food challenges for hazelnut. Allergy 2007;62(suppl. 83):105
Background: Few studies have been performed regarding how nutrient composition, for example the fat content, may affect the outcome of the allergic reaction. To test the allergenicity of products with different fat contents, patients with a lower risk of severe reactions, for example birch pollen allergic patients with oral allergy syndrome to hazelnuts, may be involved. The aim was to study if the fat content in blinded food challenges to hazelnut affects the reaction pattern in birch pollen allergic patients with reported oral allergy syndrome to hazelnut. A secondary aim was to develop 2 recipes with different fat content that was documented blind by panels using a triangle test. METHODS: Two recipes containing the same amount of hazelnut but different content of fat were developed and tested on a panel of 24 subjects with triangle tests. The fat recipe contained 28.5 % of fat and the low-fat recipe 8.5 % of fat. The recipes were tested on separate blinded food challenges on 15 patients with documented birch pollen allergy and a history of oral allergy syndrome to hazelnuts. Onset and severity of symptoms were tested using a stopwatch and a VAS-scale. RESULTS: Both recipes were regarded equal regarding taste and texture as tested by the taste panels. All 15 subjects reacted to hazelnut challenge within 7.5 minutes as expected with itching in the oral cavity and swollen feeling of tongue and lips (oral allergy syndrome), regardless of fat content. All reactions terminated within 25 minutes. Twelve out of 15 subjects reacted with a shorter onset of symptoms when challenged with the low-fat recipe (p< 0.03). For 2 subjects, the onset was shorter for the fat recipe, and one subjects reacted within the same time range for both recipes (difference less than 5 seconds). There was a wider range of reactions reported by VAS scale to the fat recipe compared to non-fat recipe. There was no correlation between severity of symptoms (measured by VAS scale) and specific IgE level to birch or hazelnut. There seems to be a delayed reaction pattern to high fat hazelnut challenges in birch pollen allergic patients. This result is in line with earlier studies on peanut challenges and may reflect the role of food composition in allergic reactions.
[89] - van der Zee JA, Wensing M, Penninks AH, Bruijnzeel-Koomen CAF, Knulst AC. Threshold levels in peanut and hazelnut allergic patients do not change in time. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°129
Introduction: Patients with food allergy frequently experience allergic reactions even though they try to avoid these foods. Most of these reactions are due to accidental ingestion of peanut or hazelnut. In a prior study we determined individual threshold doses for peanut and hazelnut in a peanut and hazelnut allergic population, which appeared to vary considerably from patient to patient. Little is known, however about the development of threshold levels in time. Objective: This study aimed at determining the course of threshold levels for peanut and hazelnut over a time period, varying from half a year to two years. Methods: Seventeen patients with hazelnut or peanut allergy that participated in a previous study were rechallenged by double-blind placebo-controlled food challenge (DBPCFC) after a period of half a year to two years. Hazelnut allergic patients (n=9) were given seven increasing doses of dried hazelnut ( 1 mg - 1 g hazelnut protein) under the same circumstances as the first challenge. Four of these patients were rechallenged twice (after half a year and a year, respectively). Peanut allergic patients (n=8) underwent a similar rechallenge with ten increasing doses of peanut meal (30 mg - 1 g peanut protein) randomly interspersed with 10 placebo meals. Results: In the hazelnut allergic population seven (out of nine) patients were rechallenged after half a year. The threshold level after half a year varied not more than a factor 3 in all of these patients as compared to the first challenge. Two hazelnut allergic patients underwent a rechallenge after one year, whereas four patients had a second rechallenge after one year. Also in this group that was rechallenged after one year, thresholds remained stable, varying not more than 3 fold, apart from one patient in which the threshold increased a factor 10. For the peanut allergic patients (n=8) rechallenges were done after a period of time varying from half a year until two years. All patients had stable threshold levels with changes not more than a factor 3 during the follow-up period. Conclusions: Threshold levels for peanut and hazelnut as determined by repeated DBPCFC remain quite stable in time. This strongly supports the value of threshold studies and facilitates the prescription of an adequate diet for the individual patient. Moreover this knowledge is important for the food industry while developing safe food for the allergic consumer.
[90] - Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-1045
BACKGROUND: Peanut and tree nut allergy are common, increasing in prevalence and the commonest food cause of anaphylaxis. In the USA, 7.8% are sensitized (have nut-specific IgE), but not all those sensitized are allergic. Lack of data makes interpretation of tests for nut-specific IgE difficult . OBJECTIVES: This is the first study to investigate the clinical significance of test results for peanut and tree nut allergy in allergic or tolerant patients. Findings are related to the severity of the allergy . METHOD: An observational study of 1000 children and adults allergic to at least one nut. History of reactions (severity graded) or tolerance to up to five nuts was obtained and skin prick test (SPT)/serum-specific IgE (CAP) performed . RESULTS: There was no correlation between SPT size and graded severity of worst reaction for all nuts combined or for peanut, hazelnut, almond and walnut. For CAP, there was no correlation for all nuts. Where patients tolerated a nut, 43% had positive SPT of 3-7 mm and 3% > or = 8 mm. For CAP, 35% were positive (0.35-14.99 kU/L) and 5% > or = 15 kU/L. In SPT range 3-7 mm, 54% were allergic and 46% were tolerant. There was poor concordance between SPT and CAP (66%). Of patients with a clear nut-allergic history, only 0.5% had negative SPT, but 22% negative CAP . CONCLUSIONS: Magnitude of SPT or CAP does not predict clinical severity, with no difference between minor urticaria and anaphylaxis. SPT is more reliable than CAP in confirming allergy. Forty-six per cent of those tolerant to a nut have positive tests > or = 3 mm (sensitized but not allergic). One cannot predict clinical reactivity from results in a wide 'grey area' of SPT 3-7 mm; 22% of negative CAPs are falsely reassuring and 40% of positive CAPs are misleading. This emphasizes the importance of the history. Understanding this is essential for accurate diagnosis. Patients with SPT > or = 8 mm and CAP > or = 15 kU/L were rarely tolerant so these levels are almost always (in > or = 95%) diagnostic. During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.
[92] - Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-1045
BACKGROUND: Peanut and tree nut allergy are common, increasing in prevalence and the commonest food cause of anaphylaxis. In the USA, 7.8% are sensitized (have nut-specific IgE), but not all those sensitized are allergic. Lack of data makes interpretation of tests for nut-specific IgE difficult . OBJECTIVES: This is the first study to investigate the clinical significance of test results for peanut and tree nut allergy in allergic or tolerant patients. Findings are related to the severity of the allergy . METHOD: An observational study of 1000 children and adults allergic to at least one nut. History of reactions (severity graded) or tolerance to up to five nuts was obtained and skin prick test (SPT)/serum-specific IgE (CAP) performed . RESULTS: There was no correlation between SPT size and graded severity of worst reaction for all nuts combined or for peanut, hazelnut, almond and walnut. For CAP, there was no correlation for all nuts. Where patients tolerated a nut, 43% had positive SPT of 3-7 mm and 3% > or = 8 mm. For CAP, 35% were positive (0.35-14.99 kU/L) and 5% > or = 15 kU/L. In SPT range 3-7 mm, 54% were allergic and 46% were tolerant. There was poor concordance between SPT and CAP (66%). Of patients with a clear nut-allergic history, only 0.5% had negative SPT, but 22% negative CAP . CONCLUSIONS: Magnitude of SPT or CAP does not predict clinical severity, with no difference between minor urticaria and anaphylaxis. SPT is more reliable than CAP in confirming allergy. Forty-six per cent of those tolerant to a nut have positive tests > or = 3 mm (sensitized but not allergic). One cannot predict clinical reactivity from results in a wide 'grey area' of SPT 3-7 mm; 22% of negative CAPs are falsely reassuring and 40% of positive CAPs are misleading. This emphasizes the importance of the history. Understanding this is essential for accurate diagnosis. Patients with SPT > or = 8 mm and CAP > or = 15 kU/L were rarely tolerant so these levels are almost always (in > or = 95%) diagnostic. During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.
[93] - Ridout S, Matthews S, Gant C, Twiselton R, Dean T, Arshad SH. The diagnosis of Brazil nut allergy using history, skin prick tests, serum specific immunoglobulin E and food challenges. Clin Exp Allergy 2006;36:226-232
BACKGROUND: Allergy to Brazil nut is a relatively common nut allergy and can be fatal. However, the evidence is lacking regarding the best approach to its diagnosis . OBJECTIVE: We sought to determine the relative merits of history, skin prick testing, measurement of serum-specific IgE and challenge in the diagnosis of Brazil nut allergy . METHODS: Fifty-six children and adults with a history of an allergic reaction to Brazil nut or evidence of sensitization were investigated by questionnaire (n=56), skin prick tests (SPTs) (n=53), measurement of serum-specific IgE to Brazil nut (n=54) and double-blind, placebo-controlled labial, and if necessary oral, challenges (n=19) . RESULTS: Brazil nut allergy occurred in highly atopic individuals of any age with a strong family history of atopy. In 24 of 56 (43%), the history of an immediate reaction was sufficient to make a diagnosis with confidence and an oral challenge was considered unsafe. Of the 19 subjects undertaking the 'gold standard' test of a double-blind, placebo-controlled, food challenge, all six subjects with a SPT of at least 6 mm had a positive challenge and all three subjects with a SPT of 0 mm had a negative challenge. In the remaining 10 (53%) subjects, where SPT was between 1 and 5 mm and serum-specific IgE was less than 3.5 kU/L, an oral challenge was performed resulting in three positive and seven negative challenges . CONCLUSION: A combination of history, SPT and serum-specific IgE was adequate in achieving a diagnosis in the majority (77%) patients with suspected Brazil nut allergy. However, a doubtful history with SPT between 1 and 5 mm, or a serum-specific IgE less than 3.5 kU/L may require an oral challenge to help determine the risk of a Brazil nut allergic reaction.
[94] - Visitsunthorn N, Tiranathanakul A, Netrakul R, Vichyanond P. Evaluation of consistency between local and imported seafood allergen extracts. J Med Assoc Thai 2002;85(Suppl. 2):S593-S598
BACKGROUND: Seafood is a common cause of food allergy in Thai adults and children. Skin prick test is a safe and convenient way to screen seafood allergy. To date, the Allergy Unit, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University uses imported extracts for seafood skin prick test. The extracts are expensive and may not be the same species as seafood in Thailand. OBJECTIVES: To compare the consistency between local seafood allergen extracts prepared by the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University and imported seafood allergen extracts. METHODS: The study was a prospective comparative trial performed in children who attended the Pediatric Allergy Clinic, Siriraj Hospital from March 1999 to October 2000. The skin prick test was performed with the local seafood allergen extracts prepared by a pharmacist from the Department of Pharmacology and the imported seafood allergen extracts included shrimp, fish and crab. Histamine and normal saline were used as positive and negative control respectively. The positive result was recorded when wheal reaction was > or = 3 mm larger than negative control. RESULTS: Eighty eight patients (57 boys and 31 girls) were included in this study. The average age was 7.7 years (1-15 years). Half of the patients had a history of seafood allergy. The study showed probable consistency between imported and local skin prick test of shrimp and crab extracts (kappa = 5-7) but no consistency between imported and local skin prick test of fish extracts (kappa < 5). The study also showed no consistency between history of seafood and skin prick test result. CONCLUSION: Local seafood allergen extracts from the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University are safe but they cannot replace the imported seafood allergen extracts. Further investigations about sensitivity and specificity of both kinds of allergen extracts are necessary.
[95] - 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"
[97] - Flinterman AE, Akkerdaas JH, den Hartog Jager CF, Rigby NM, Fernandez-Rivas M, Hoekstra MO, et al. Lipid transfer protein–linked hazelnut allergy in children from a non-Mediterranean birch-endemic area. J Allergy Clin Immunol 2008;121:423-428
BACKGROUND: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8 . OBJECTIVE: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area . METHODS: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed . RESULTS: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1 . CONCLUSION: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.
[99] - Flinterman AE, Akkerdaas JH, den Hartog Jager CF, Rigby NM, Fernandez-Rivas M, Hoekstra MO, et al. Lipid transfer protein–linked hazelnut allergy in children from a non-Mediterranean birch-endemic area. J Allergy Clin Immunol 2008;121:423-428
BACKGROUND: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8 . OBJECTIVE: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area . METHODS: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed . RESULTS: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1 . CONCLUSION: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.
[100] - Malandain H. IgE-reactive carbohydrate epitopes--classification, cross-reactivity, and clinical impact. Eur Ann Allergy Clin Immunol 2005;37:122-128
A glycan-related IgE-reactivity has been demonstrated in most allergen sources, especially in plant kingdom. Recent progress in glycobiology has allowed a clearer classification of these glyco-epitopes. Unlike classical peptide chain-based epitopes, glyco-epitopes can share significant structural homologies beyond the limits of protein families. These glycoepitopes are thus prone to extensive cross-reactivity. They have been called Cross-reactive Carbohydrate Determinants or CCD. Many of these glyco-epitopes behave as "panepitopes", leading to cross-reactivity between products as distant as pollens and hymenoptera venoms. But CCD are not universally cross-reactive and they rather cluster into subgroups such as plant CCD or fungal CCD. Because a monovalent IgE-binding is sufficient in serum-based assays, glyco-epitopes and CCD are classically considered as a potential source of positive in vitro results without clinical significance. But some authors recently demonstrated that glyco-epitopes could induce a response at the cell level and suggested that they might play a role in vivo. As long as in vitro assays include glycan- and CCD-related IgE responses, laboratory results should be carefully interpreted in the light of existing knowledge about the glycomes of natural products. IgE-reactivity of the patient's serum can also be tested towards a glycoprotein model such as bromelain.
[101] - Poltronieri P, Cappello MS, Dohmae N, Conti A, Fortunato D, Pastorello EA, et al. Identification and Characterisation of the IgE-Binding Proteins 2S Albumin and Conglutin gamma in Almond (Prunus dulcis) Seeds. Int Arch Allergy Immunol 2002;128:97-104
Background: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. Methods: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. Results: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. Conclusions: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.
[102] - Garcia RN, Arocena RV, Laurena AC, Tecson-Mendoza EM. 11S and 7S globulins of coconut (Cocos nucifera L.): purification and characterization. J Agric Food Chem 2005;53:1734-1739
Total globulins extracted with 0.4 M NaCl in buffer from coconut endosperm separated into two peaks on gel filtration: peak I corresponding to 11S globulin or cocosin and peak II to 7S globulin with native molecular weights of 326 000 and 156 000, respectively. The percent composition of total globulins was estimated to be 11S, 86% and 7S, 14%. On SDS-PAGE, cocosin resolved into two closely migrating bands at approximately 34 000 (acidic polypeptide) and another set of 2 bands at 24 000 (basic polypeptide). Each set consisted of one darkly stained band and one lightly stained band. The 7S globulin consisted of three bands of 16 000, 22 000, and 24 000. Three isoforms of cocosin were identified after anion exchange chromatography. Cocosin, but not the 7S, was found to have disulfide bonds. Using periodic acid-Schiff's reagent, all of the bands of cocosin on SDS-PAGE were positive for carbohydrate. However, when con A-peroxidase was used, only the basic polypeptide stained positively for carbohydrate. For the 7S globulin, no carbohydrate group was detected using the PAS and con A-peroxidase tests. The 7S globulin was easily extracted with 0.10-0.15 M NaCl, whereas cocosin was extracted with 0.35 M NaCl. The N-terminal amino acid sequences of the 34 k band and 24 k band of cocosin were SVRSVNEFRXE and GLEETQ, respectively, and that of the 7S was EQEDPELQK.
[103] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334
In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.
[104] - Wilson IBH, Harthill JE, Mullin NP, Ashford DA, Altmann F. Core alpha-1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 1998;8:651-661
Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.
[105] - Wilson IBH, Zeleny R, Kolarich D, Staudacher E, Stroop CJM, Kamerling JP, et al. Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha-1,3-linked fucose and xylose substitutions. Glycobiology 2001;11:261-274
The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.
[106] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220
BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.
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