Accueil / Information / Aller plus loin... / Aériens / Environnement intérieur / Les blattes
Les blattes
lundi 16 mars 2009, par
Seules quelques espèces de blattes intéressent l’allergologie 
.
Blatella germanica est un insecte purement domestique. Il est retrouvé dans toutes les régions du monde.
Periplaneta americana est très répandu aussi, mais dans des pays à climat plus chaud/humide.
- On trouve P. americana dans les maisons mais aussi dans des bâtiments publics.
- P. americana peut cohabiter avec B. germanica, l’exposition aux allergènes étant alors double.
P. americana est plus répandue que B. germanica au Japon, à Taïwan, au Brésil, à Singapour, en Malaisie.
Par contre en Europe, aux USA, en Corée B. germanica prédomine.
Periplaneta japonica est répandu en Corée, de même que Periplaneta fuliginosa au Japon. Blatella orientalis peut être vue en Europe. Supella Pongipalpis est plutôt présente dans les pays tropicaux/subtropicaux.
Les infestations de blattes indoor sont nettement reliées aux conditions d’habitation, lesquelles sont souvent dépendantes de facteurs socio-économiques .
Des débris de blattes peuvent contaminer des farines , mais, contrairement aux acariens retrouvés dans des farines, il ne semble pas avoir été rapporté d’allergie alimentaire aux blattes.
Un asthme du boulanger du aux blattes est, lui, décrit .
Les allergènes des blattes
Les allergènes de blattes proviennent aussi bien de leurs excréments que des débris d’insectes. Notamment, le nombre élevé de mues entre le stade nymphal et le stade adulte (6 à 12) provoque la formation de débris d’exosquelette et d’exuvies.
Certains auteurs pensent que les excréments seraient plus allergisants .
Les allergènes sont portés par des particules dans la poussière. Celles-ci ont des tailles variables, souvent grosses et sédimentant facilement, mais parfois petites et aptes à atteindre les bronchioles. Les allergènes de blattes sont très asthmogènes (cf. blattes et activité biochimique).
Les allergènes semblent pouvoir être transportés par des vêtements (écoles) dans des cas de contamination importante.
On n’a caractérisé qu’une minorité des allergènes des blattes. Par exemple, pas moins de 12 spots IgE-réactifs en plus de ceux de Per a 1 et Per a 7 sont révélés en immunoblot 2D avec P. americana .
Les allergènes de blatte ne dérogent pas à la règle et se présentent souvent sous plusieurs isoformes et sont capables de dimériser (ex Bla g 2 ) ou de se fragmenter (ex Bla g 1 ), ce qui rend complexe l’interprétation des immuno-empreintes de leurs extraits.
B. germanica et P. americana principalement ont été étudiées. A l’image des acariens, des "groupes" d’allergènes communs à différentes blattes ont été définis.
Par exemple le groupe 7 comprend des tropomyosines.
Cependant, beaucoup d’allergènes ont une IgE-réactivité mal établie, même si ces protéines ont été acceptées par l’IUIS.
Allergènes identifiés dans les différentes blattes
| Groupe | Fonction | B.germanica | B.orientalis | P.americana | P.fuliginosa |
|---|---|---|---|---|---|
| 1 | ? | IUIS | ? | IUIS | 0 |
| 2 | Aspartate protéases | IUIS | 0 | ||
| 3 | Arylphorines | IUIS | |||
| 4 | Lipocalines | IUIS | 0 | ||
| 5 | Glutathion-S-transférases | IUIS | 0 | ||
| 6 | Troponines C | IUIS | IUIS | ||
| 7 | Tropomyosines | IUIS | ? | IUIS | 0 |
| 8 | Chaîne légère de myosines | IUIS | |||
| 9 | Arginine kinases | 0 | IUIS | ||
| 10 | Sérine protéases | 0 | IUIS |
Quels sont les allergènes importants ?
Le groupe 1 (Bla g 1, Per a 1) :
Il n’a pas été trouvé de fonction biochimique pour ces allergènes jusqu’à présent.
Une petite homologie (27-32% ) avec une protéine ANG12 du moustique Anopheles gambiae et, comme cette dernière, Bla g 1 et Per a 1 auraient une origine intestinale.
De fait, on a montré la présence de Bla g 1 dans les excréments de blatte et la production de Bla g 1 semble varier parallèlement à la prise alimentaire.
Les femelles produisent environ 6 fois plus de Bla g 1 que les mâles et environ 30 fois plus que les nymphes .
Bla g 1 et Per a 1 sont formés de 2 duplex d’acides aminés similaires. Ils ont de 77 à 85 % d’identité entre eux, selon leurs différentes isoformes .
Certains auteurs estiment que les réactions croisées entre B. germanica et P. americana sont essentiellement dues à Bla g 1 et Per a 1 .
Ce sont les concentrations de Bla g 1/Per a 1 par gramme de poussière qui servent à définir les seuils sensibilisants (2 U/g) et asthmogène (8 U/g) pour l’exposition indoor aux blattes.
On estime à 25-50 % la prévalence de positivité pour Bla g 1 chez les sujets allergiques à B. germanica. Elle est plus élevée souvent pour Per a 1 : de 54 à 77%, selon les isoformes .
Le groupe 2 (Bla g 2, Per a 2) :
Ces allergènes lient le zinc et ont une structure les classant dans les aspartate protéases. Cependant ils n’ont pas (ou extrêmement peu ) d’activité protéasique.
Per a 2 n’a été que récemment découvert. Le % d’identité entre Bla g 2 et Per a 2 est faible (44 %) .
Bla g 2 est puissamment allergisant .
Son origine dans la blatte est probablement digestive.
De 55 à 80 % des sujets allergiques à B. germanica sont positifs pour Bla g 2 .
Les réactions croisées de Bla g 2 avec des aspartate protéases d’autres organismes sont à exclure car les % d’identité sont trop faibles .
Le groupe 3 :
On n’a pas trouvé d’équivalent à Per a 3 dans B. germanica pour le moment. Per a 3 a une origine digestive .
La fonction biochimique de Per a 3 est inconnue : une petite homologie (20-36%) a été notée avec des protéines d’hémolymphe d’insectes (ex arylphorines, hémocyanines) .
Per a 3 se présente sous la forme d’hexamères, lesquels montrent une activité dégranulante plus grande que les monomères .
Per a 3 possède des variants très différents les uns des autres (de 46 à 75 kD) : en tests cutanés ils ont une réactivité très différente aussi, de sorte que la prévalence de positivité pour Per a 3 va de 27 à 95 % parmi des sujets allergiques à P. americana .
Le groupe 4 :
Bla g 4 est une lipocaline produite par les blattes mâles.
Prévalence de positivité 20-60 %.
Pas de réactivité croisée avec d’autres lipocalines (chien, lait, etc…) du fait du trop faible niveau d’identité avec celles-ci (19-24 %) .
Le groupe 5 :
Bla g 5 appartient à la famille des glutathion-S-transférases (GST). Ces enzymes sont très hétérogènes et l’on les distingue en plusieurs classes de GST : Bla g 5 est une sigma GST.
Avec Bla g 2, Bla g 5 est souvent présenté comme parmi les allergènes les plus importants dans B. germanica. Cependant, la prévalence de positivité est assez variable d’unpays à un autre (20 à 70 %) .
Du fait de la fréquence des double réactivités acarien/blatte, la présence d’une GST dans D. pteronyssinus a soulevé la question d’une réaction croisée acarien/blatte par le biais de GST.
Cependant le pourcentatge d’identité entre Bla g 5 et Der p 8 n’est que de 27 % . Il semble peu probable qu’une réactivité croisée ait lieu entre ces 2 allergènes .
Le groupe 6 :
Ce sont des troponines C. Selon les isoformes, l’identité entre Bla g 6 et Per a 6 varie de 69 à 91%. L’IgE-réactivité de ces allergènes est moindre en l’absence de calcium (ex. dans les extrait ?) . Peu de données sur la prévalence de positivité (14% ).
Le groupe 7 :
Il comprend les tropomyosines des blattes.
Des réactions croisées à l’intérieur du groupe 7 sont à prévoir (plus de 95 % d’identité). Elles peuvent s’étendre à des tropomyosines de crustacés .
Pour les acariens, des résultats peu probants ont été obtenus :
- D. pteronyssinus ou D. farinae inhibent B. germanica ou P. fuliginosa ; mais la tropomyosine Per f 7 n’inhibe D. farinae que partiellement (cf. Blattes et acariens).
La prévalence de positivité pour les tropomyosines de blattes est de 10-15 % en Europe et aux USA alors qu’elle est de 50-55 % en Afrique et au Brésil. Les helminthes ayant des tropomyosines allergisantes, la relation parasitose/sensibilisation aux blattes a été soulevée (cf. Blattes et parasitose) .
Le groupe 8 :
Une sous-unité de myosine est IgE-réactive dans B. germanica. Dénommé Bla g 8, cet allergène présente une homologie modérée avec Lit v 3 qui est aussi une chaîne légère de myosine dans la crevette Litopenaeus vannamei (une crevette produite notamment en aquaculture) .
Le groupe 9 :
Ce sont des arginine kinases et donc des protéines musculaires comme le groupe 7 et le groupe 8. La réactivité croisée des arginine kinases de blattes avec celles de crevette n’a été montrée à l’heure actuelle qu’avec un anticorps monoclonal (pas avec des IgE de patients).
Néanmoins, les arginine kinases sont, comme les tropomyosines, fortement homologues (ex. 97% d’identité entre Per a 9 et Bla g 9 ) et seraient de bons candidats à une réactivité croisée balttes-crevettes, voire plus généralement à participer à un groupe de panallergènes d’arthropodes .
Le groupe 10 :
Des sérines protéases ont montré leur IgE-réactivité dans P. americana et B. germanica. Elles ont une homologie modérée avec les sérine protéases composant le groupe 3 des acariens (42-57% d’identité ). Mais elles semblent mieux résister que ces dernières à la chaleur .
Autres protéines IgE-réactives :
Une seconde glutathion-S transférase, de la classe delta cette fois, a été montrée IgE-réactive chez environ 15% des patients. Cette enzyme de B. germanica n’a, en fait, que 15% d’identité avec l’autre GST (Bla g 5) et ne croise pas avec elle .
Une troponine T (et non C comme celles du groupe 6) a été trouvée IgE-réactive chez 17% de patients allergiques à P. americana .
On le voit, le catalogue des allergènes de blattes est large. Comme pour d’autres arthropodes dont l’allergénicité se manifeste par inhalation de protéines aérosolisées, ce sont souvent des particules émanant de débris de blattes qui portent les allergènes. Il n’est donc pas surprenant que, parmi les allergènes de blattes, on retrouve nombre de protéines participant à la fonction musculaire : tropomyosines, troponines, arginine kinases.
Blattes et activité pro-inflammatoire des allergènes
On sait à présent que si les allergènes du groupe 2 des blattes (ex. Bla g 2) sont à peu près dépourvus d’activité protéasique, d’autres protéines sont des protéases et pourraient jouer un rôle similaire à celui remarqué pour les acariens.
En gel bidimensionnel, 10 spots ont une activité protéasique dans un extrait de B. germanica . Même si 1 seul d’entre eux correspond à un allergène connu, la possibilité d’une action pro-inflammatoire des poussières inhalées de blatte est réelle.
De fait, la sérine protéase isolée de B. germanica par l’équipe de Pomés n’est pas IgE réactive.
Plusieurs travaux ont montré in vitro une action des extraits de blatte sur le récepteur PAR-2 avec activation des éosinophiles , libération d’IL8 , augmentation de la perméabilité épithéliale bronchique ou action sur le remodelage des voies aériennes .
Cette activité pro-inflammatoire pourrait contribuer au pouvoir asthmogène de l’exposition aux poussières de blattes.
Blattes et acariens
Les sujets positifs pour B. germanica sont très souvent positifs aussi pour D. pteronyssinus . Il en est de même pour P. fuliginosa et D. farinae au Japon .
La fréquence des tests cutanés positifs pour D. pteronyssinus a été trouvée doublée en Scandinavie (36 % vs 17 %) chez les sujets qui étaient positifs en TC pour B. germanica .
En Croatie, des TC+ pour B. germanica étaient très souvent accompagnés de TC positifs pour des acariens de stockage . Cette relation entre acariens de stockage et positivité pour acariens domestiques et/ou blattes a aussi été évoquée en Islande .
Face a un résultat positif pour une blatte et pour un acarien, la question est de savoir si cela provient d’une double sensibilisation ou d’une seule sensibilisation à laquelle s’ajoute une réactivité croisée. Dans le cas d’espèce, ce pourraient être les acariens qui induisent une réactivité aux blattes. Ainsi :
- 19% d’allergiques aux acariens présentaient un TC positif pour B. germanica dans une étude menée au Portugal
- et plusieurs travaux ont montrait que D. pteronyssinus inhibait B. germanica ou que D. farinae inhibait P. fuliginosa . Dans la mesure où l’inhibition inverse était plus difficile, cette dissymétrie pourrait indiquer l’initiateur de la sensibilisation, donc ici les acariens.
Cependant, la présence de blattes dans la maison peut contribuer à augmenter la fréquence des TC positifs pour D. pteronyssinus :par exemple, 30 % vs 20 % en l’absence de blatte .
Aussi, Pomés et Sidenius favorisent l’hypothèse d’une double exposition et d’une double sensibilisation. Et Arruda estime que la relevance clinique d’une relation acariens-blattes n’est pas démontrée .
Cela n’exclut pas une part de réactivité croisée.
Les tropomyosines sont les allergènes qui ont le plus de chances d’être responsables de ces RC du fait des bons pourcentages d’identité entre les tropomyosines :
- 25 % des sujets positifs pour P. americana et D. pteronyssinus (USA) étaient positifs pour rPer a 7.Et une bonne corrélation a été notée entre rPer a 7 et rDer p 10 (r = 0,90) .
- dans une série de 13 patients Martiniquais, avec asthme/rhinite et allergie aux crustacés, 10 sujets étaient positifs pour Der p10 et cinq pour Bla g 7 .
- dans une étude en Louisiane portant sur 54 patients allergiques aux crevettes, il a été relevé 77% de TC positifs pour les acariens et 56% pour la blatte .
On voit que là où une exposition significative aux acariens et aux blattes s’accompagne d’une alimentation riche en Crustacés, il sera bien difficile de démêler réactivité croisée à des tropomyosines et sensibilisation réelle aux blattes. Et ce problème se complique encore dans les zones d’endémie de parasitose.
Blattes et parasitoses
Plusieurs études ont montré un décalage important de positivité entre Europe/USA et Afrique/Amérique du sud pour les tropomyosines de blattes.
Sachant que les tropomyosines d’ascaris sont relativement homologues (69-74 % d’identité) de celles de blattes, la question d’un effet de la parasitose sur la réactivité et/ou l’allergie aux blattes a été soulevée.
Chez des sujets Brésiliens avec asthme/rhinite, la mesure in vitro de différents recombinants de P. americana a montré que rPer a 7 (tropomyosine) est, de loin, l’allergène le plus souvent positif (42 % des sujets).
Dans le même temps la tropomyosine d’ascaris était positive chez 51 % des sujets .
Des résultats similaires ont été notés dans d’autres cohortes , avec une bonne corrélation entre les résultats in vitro pour Bla g 7 ou Per a 7 et ceux pour la tropomyosine d’ascaris.
La fréquente positivité pour une tropomyosine de blatte (Bla g 7 et/ou Per a 7) est-elle la simple conséquence d’une réaction croisée avec la tropomyosine d’ascaris en zone d’endémie ?
Arruda retient la possibilité d’une sensibilisation aux blattes favorisée, chez certains sujets, par une sensibilisation initiale aux tropomyosines parasitaires . Mais l’incidence ou la sévérité de l’asthme n’était pas corrélée avec le degré de réactivité aux tropomyosines dans une étude d’enfants Brésiliens .
Blattes et difficultés diagnostiques
Les extraits de blattes ne sont pas parfaits.
Leurs contenus sont variables d’un fabricant à un autre et ils sont susceptibles de se dégrader du fait des protéases .
Il a été noté aussi qu’il fallait veiller à éviter les contaminations des matières premières par des acariens présents dans les cultures de blattes .
Enfin, il est possible que l’extraction des allergènes soit incomplète : en partant de blattes lyophilisées, on a pu montrer un nombre plus élevé de bandes IgE réactives que dans les extraits commerciaux (22 au lieu de 6-11) .
Des essais préliminaires ont été entrepris pour valider l’utilisation de recombinants de blatte chez des enfants Brésiliens : rBla g 2, g 4, et g 5 et rPer a 7 (mais pas rPer a 1) étaient significativement associés à un wheezing ; de meilleurs résultats étaient cependant obtenus avec les recombinants de D. pteronyssinus . Des progrès restent donc à faire.
Blattes et CCD
(voir aussi : Les CCD)
Peu de travaux ont abordé la question :
- un anticorps monoclonal a détecté une N-glycosylation sur Bla g 2 . Mais cela ne signifie pas que cette dernière soit IgE-réactive chez l’Homme.
- pourtant, à partir d’une banque de sérum, des auteurs suédois ont montré que les sujets présentant un résultat pour la blatte germanique supérieur à celui pour D. pteronyssinus et la crevette étaient très souvent positif pour le test MUXF3 (glyco-épitopes de broméline) .
- Un sérum ayant 65 kU/l pour la blatte germanique et > 100 kU/l en MUXF3 a été étudié. Ce sérum n’était pas positivé par Bla g 7 (tropomyosine) puisqu’une inhibition par rPen a 1 ne montrait aucune baisse du résultat pour la blatte.
- Par contre, la broméline faisait chuter de 98 % le résultat de la blatte.
- Et de même, chez un patient pollinique, allergique aux acariens et à l’abeille, le pollen de bouleau et l’abeille (mais pas D. pteronyssinus) inhibaient B. germanica
Des résultats similaires ont été obtenus avec une technique permettant de capturer les IgE anti-glycanes .
Il paraît donc recevable de considérer que les IgE anti-CCD de végétaux et/ou d’Hyménoptères peuvent parfois perturber les tests in vitro pour les blattes.
[1] - Pomés A. Cockroach and other inhalant insect allergens. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 183-200 ↩
[2] - Helm RM, Pomes A. Cockroach and other inhalant insect allergens. Clin Allergy Immunol 2004;18:271-296 ↩
[3] - Arruda LK. Cockroach allergens. Curr Allergy Asthma Rep 2005;5:411-416
Cockroaches produce several potent allergens associated with sensitization and the development of asthma. Many of these allergens have been cloned and produced as biologically active recombinant proteins. Three-dimensional structure and molecular modeling, in addition to studies on the biological functions of these proteins in the cockroach, have provided new insights into the ability of these allergens to cause immunoglobulin E antibody responses. Recent studies have pointed to a potential role of recombinant cockroach allergens for diagnosis and the development of novel therapeutic strategies that may benefit cockroach-allergic patients.
↩[4] - de Blay F, Casel S, Pauli G, Bessot JC. Allergies respiratoires et environnement allergénique domestique. Rev Mal Respir 2000;17:167-176
Based on immunoassay techniques used to identify and quantify major indoor allergens, epidemiological studies have demonstrated that level of exposure depends on the geographical situation (climate, urbanization) and that there is a relationship between exposure and sensitization of predisposed patients. Most of the major mite allergens are proteases. For mammals and cockroaches, the major allergens belong to the retinal protein binding group. Determination of the airborne characteristics of the major indoor allergens is helpful in better understanding the pathophysiology of asthma. In the future, more precise diagnosis and more efficient immunotherapy will be possible through the development of recombinant allergens. The efficacy of allergen avoidance in allergic asthma has been demonstrated in several studies. Thus the respective role of different household allergens should lead to improved diagnosis and treatment of allergic diseases.
↩[5] - de Blay F, Lieutier-Colas F. Epidémiologie de l'allergie aux blattes et caractéristiques de l'exposition aux allergènes des blattes. Rev Fr Allergol Immunol Clin 1998;38:839-841 ↩
[6] - Gore JC, Schal C. Cockroach allergen biology and mitigation in the indoor environment. Annu Rev Entomol 2007;52:439-463
For nearly a half century, cockroaches have been recognized as a major cause of asthma morbidity in the urban, inner-city environment. Several cockroach-produced allergens have been identified and characterized, and a few have been produced as recombinant proteins. Recent research has moved beyond clinical, patient-based investigations to a more entomological perspective that addresses the production, physiological regulation, and developmental expression of cockroach allergens, thus providing insight into their functional biology and their relationship to current cockroach control strategies. Although successful removal of cockroach allergens from the infested environment has been difficult to accomplish with remedial sanitation, large-scale reductions in cockroach allergens below clinically relevant thresholds have recently been realized through suppression of cockroach populations. Here we review the current understanding of cockroach allergen biology and the demographics associated with human exposure and sensitization. We also critically evaluate allergen mitigation studies from an entomological perspective, highlighting disparities between successful and failed attempts to lessen the cockroach allergen burden in homes.
↩[8] - Marraccini P, Previdi M, Cantone L, Varin E, Salimbeni R, Todaro A, et al. The possible role of cockroaches in baker's asthma. Med Lav 2007;98:284-288
BACKGROUND: Baker's asthma is related to wheat flour exposure and to other cereal dust exposure. OBJECTIVES: The cockroach is considered a significant allergen and can occasionally trigger asthma in bakery workers.
METHODS: The case of a 33-year-old male, suffering from asthma in the workplace with previous equivocal tests for cereal dust was investigated Clinical assessment of the worker consisted of cutaneous and blood screening for common and occupational allergens, including cockroach. The subject was monitored for aspecific bronchial reactivity and peak flow in a cockroach disinfected workplace, and these data were compared to data obtained after previous workplace exposure.
RESULTS: The worker was not allergic to wheat and other cereal dusts or alpha-amylase, but was sensitized to cockroach. His asthmatic symptoms disappeared, and bronchial reactivity varied after a long period outside the bakery workplace. PEF monitoring, that had showed diurnal variability > 20% and differences between working and non-working periods, demonstrated both normal values and daily variations less than 10% when he returned to the cockroach disinfected workplace.
CONCLUSIONS: The cockroach is a common allergen, however no case of work-related baker's asthma due to the cockroach has been previously described. Clinical history and analysis of the allergens at the workplace must direct the clinical approach of the investigators, in order to correctly evaluate the subject and enable him/her to resume work.
↩[9] - Pomés A, Wünschmann S, Hindley J, Vailes LD, Chapman MD. Cockroach allergens: function, structure and allergenicity. Protein Pept Lett 2007;14:960-969
Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.
↩[10] - Yun YY, Ko SH, Park JW, Lee IY, Ree HI, Hong CS. Comparison of allergenic components between German cockroach whole body and fecal extracts. Ann Allergy Asthma Immunol 2001;86:551-556
BACKGROUND: Cockroaches have been demonstrated to be an etiologic factor in allergic diseases. Further, sensitivity to cockroach places patients with asthma at risk for exacerbations that require emergency medical care. OBJECTIVE: This study compared the differences in allergenic components between German cockroach whole body and German cockroach fecal extracts (GWBE and GFE).
METHODS: Patients with asthma and/or allergic rhinitis were skin prick tested with German cockroach extract (Bayer Corporation, West Haven, CT). Serum specimens from these patients, 25 with positive skin tests and 8 with negative tests, were used for the ELISA and immunoblot experiments.
RESULTS: By ELISA, 72% (18 of 25) and 60% (15 of 25) of positive responders' sera showed IgE antibodies to GWBE and GFE, respectively, and the IgE levels to GWBE were highly correlated with those to GFE (r = .84, P < .01). In inhibition ELISA experiments, extensive cross-reactivity was observed between GWBE and GFE, slight cross-reactivity between GWBE and Dermatophagoides farinae, and no cross-reactivity between GFE and D. farinae. The two-site monoclonal antibody ELISA detected more of the German cockroach major allergens in GFE compared with GWBE; 6.2 times (2420 vs 390 U/mL) for Bla g 1 and 3 times (15.32 vs 5.07 microg/mL) for Bla g 2. In the immunoblot comparison of patients' sera, the IgE antibodies binding to GWBE were apparently different from those binding to GFE in all the positive responders' sera; eg, 50% or more of the 25 positive responders' sera reacted to 43- to 67-kDa proteins in GWBE and to 28- to 30-kDa proteins in GFE, respectively. No IgE antibodies bound to components in GWBE and GFE in the 8 negative responders' sera.
CONCLUSIONS: There are major differences between the allergenic components of GWBE and GFE. Based on the amounts of major allergens (Bla g 1, Bla g 2), German cockroach feces are a more important source of allergen than the whole body in respiratory allergic diseases.
↩[11] - Barbosa MCR, Santos ABR, Santos LD, Palma MS, Arruda LK. Use of Proteomic Analysis for Identification of Novel Cockroach Allergens. J Allergy Clin Immunol 2009;123:S98
RATIONALE: Cockroach allergy is a major cause of asthma.
Approximately 50% of cockroach-allergic patients with asthma and/or rhinitis living in Brazil, identified by skin testing with commercial cockroach extracts, show IgE sensitization to Periplaneta americana allergens Per a 7 (tropomyosin) (42%) and/or Per a 1 (5%). It would be useful to identify new P. americana allergens to improve diagnosis and treatment of cockroach allergy.
METHODS: Cockroach proteins detected by 2-D electrophoresis were visualized by Coomassie Brilliant Blue staining, and were transferred to nitrocellulose membrane. Immunoblotting was carried out using undiluted serum pool constructed with sera obtained from 27 patients with asthma and/or rhinitis allergic to P. americana and with negative skin tests and serum IgE to Per a 7 and Per a 1. The membrane was incubated with biotinconjugated anti-human IgE (1:250), followed by Streptavidin Horseradish Peroxidase. Detection was performed by Enhanced Chemiluminescence (ECL) with exposure of 30 minutes.
RESULTS: The proteomic study of P. americana extract showed approximately 100 spots with pI ranging from 3 to 9, and molecular mass varying from 14 to 60 kDa. Of these proteins, 12 showed IgE reactivity using the cockroach-allergic serum pool. These IgE-binding proteins had pI 3 to 5 and molecular mass of 45 to 50 kDa.
CONCLUSIONS: Proteomic analysis P. americana extract can provide novel information on cockroach allergens and related isoforms, which could contribute to better diagnosis and treatment of cockroach allergy.
↩[12] - Pomés A, Li M, Wünschmann S, Chapman MD, Wlodawer A, Gustchina A. Cockroach Allergen Bla g 2 Dimerizes in a Crystal Complex with an Antibody Fragment. J Allergy Clin Immunol 2007;119(1 suppl):S104
RATIONALE: Bla g 2 is a potent German cockroach allergen, homologous to aspartic proteases. The aim was to study the antigenic determinants of the allergen.
METHODS: Purified recombinant Bla g 2 was co-crystallized with Fab' fragments of the monoclonal antibody (mAb) 7C11 obtained by pepsin cleavage. Crystals grew in mother liquid containing 16% PEG10k, 0.2M Ammonium Sulfate and 5mM DTT, pH 7.0. The structure was solved by molecular replacement and refined using the amino acid sequence obtained by Reverse-Transcription-PCR of mRNA from the cell line producing 7C11 mAb.
RESULTS: Bla g 2 is present in the complex in a dimeric state, unusual for the aspartic protease family. Interactions with the antibody are dominated by a number of charged residues of the allergen. Two Fab' molecules interact with an allergen dimer that is stabilized by the formation of a four-helical bundle comprised of two a-helices from each allergen monomer. This novel dimerization mode is attributed to the presence of a unique di-sulfide bridge between C51a and C113, connecting two helices within each Blag 2 monomer. Two mutants of either the two cysteines alone, or both cysteines plus the residues N52, Q110 and I114 that may be involved in dimerization, were expressed in Pichia pastoris. These mutants will allow investigating the importance of Blag 2 dimerization on allergenicity.
CONCLUSIONS: Determination of the molecular structure of a Bla g 2-Fab' complex enables a rational approach to study the effect of dimerization on allergenicity and facilitates epitope mapping and engineering of allergen molecules with reduced IgE antibody binding.
↩[13] - Helm RM, Pomes A. Cockroach and other inhalant insect allergens. Clin Allergy Immunol 2004;18:271-296 ↩
[14] - Jeong KY, Hong CS, Yong TS. Recombinant allergens for diagnosis and immunotherapy of allergic disorders, with emphasis on cockroach allergy. Curr Protein Pept Sci 2006;7:57-71
The prevalence of allergic disorders has increased over the past few decades and the quality of life has been significantly influenced at least for the allergic subjects. Allergen avoidance is thought to be the best way of preventing clinical manifestation of the disease, however, it is not possible for some allergens, and other pharmacological and/or immunological treatment has to be made. Repetitive injection of sensitized allergens to the patients (immunotherapy) is the only known curative approach to the disease even though the exact mechanism is not clear to date. Crude extract of allergens has lots of shortcomings which might arouse unexpected results. Genetic engineering and recombinant allergens are thought to be one of the alternative ways to overcome these limitations. Genetic engineering could facilitate the investigation of immune responses of the subjects especially on B cell and T cell epitopes, and produce the therapeutic allergens which might minimize the possible side effects. Furthermore, conjugation of immuno-modulatory molecules such as CpG-ODN, cytokines, or toxins which could act specifically to the given allergens, and maleylation of the allergens could maximize the prophylactic or therapeutic effect. Immunotherapies for the pollen allergy and insect sting allergy have been thought to be successful. House dust mite allergy and cockroach allergy have been reported less beneficial by immunotherapeutic approaches. Cockroaches are one of the most important causes of asthma, and severe complications are often reported in the children in city dwellers with low-incomes. The studies of the biological functions of cockroach allergens and the use of recombinant allergens should allow understanding of mechanisms of cockroach-elicited allergic disorders and development of allergen-specific and sensitive diagnostics and tailored therapeutic approaches in the future.
↩[15] - Wang NM, Lee MF, Wu CH. Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen. Allergy 1999;54:119-127
BACKGROUND: Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties.
METHODS: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE.
RESULTS: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino- acid protein (50kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42.
CONCLUSIONS: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE- reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies
↩[16] - Gore JC, Schal C. Expression, production and excretion of Bla g 1, a major human allergen, in relation to food intake in the German cockroach, Blattella germanica. Med Vet Entomol 2005;19:127-134
The German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), produces several potent human allergens, one of which, Blattella germanica allergen 1 (Bla g 1), is produced in the midgut and excreted in faeces. We tested with descriptive and experimental approaches the hypothesis that Bla g 1 production is related to food intake in adult males and females of the German cockroach. Bla g 1 mRNA expression in the female midgut (assayed by real time quantitative polymerase chain reaction), her Bla g 1 content (assayed by enzyme-linked immunosorbent assay), and the female's faeces production and its Bla g 1 content tracked a cyclic pattern in relation to the gonadotrophic cycle. All four measures rose as food intake increased, declined before oviposition in relation to diminishing food intake, and remained low while the female carried an egg case for 20 days. After her first clutch of embryos hatched, the female resumed feeding, and faeces and Bla g 1 production increased concomitantly. Both Bla g 1 mRNA expression and Bla g 1 protein levels remained low in experimentally starved females. However, when starved females were allowed to feed, Bla g 1 production elevated and the gonadotrophic cycle resumed. Bla g 1 mRNA expression also increased six-fold in response to feeding compared to starved females. By contrast, there were no apparent cycles in the pattern of Bla g 1 production in males, reflecting their low and non-cyclic food intake. Our results therefore demonstrate that Bla g 1 production in B. germanica is modulated in relation to food intake.
↩[17] - Wang NM, Lee MF, Wu CH. Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen. Allergy 1999;54:119-127
BACKGROUND: Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties.
METHODS: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE.
RESULTS: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino- acid protein (50kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42.
CONCLUSIONS: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE- reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies
↩[18] - Katial RK. Cockroach allergy. Immunol Allergy Clin North Am 2003;23:483-499
The ubiquitous existence of cockroaches and the large-scale domestic infestation seen in inner cities make cockroach proteins a significant indoor allergen and a risk factor for asthma among inner-city residents. Studies have shown that early exposure to high levels of allergen may lead to the development of asthma in individuals with a genetic predisposition to asthma. Although field trials at cockroach abatement do not yield promising results, integrated pest management still remains the best control strategy. In highly susceptible or symptomatic patients, allergen-specific immunotherapy may be beneficial, although data are limited. As molecular techniques improve and recombinant allergens are developed, a more novel form of T-cell-specific immunotherapy may prove to be efficacious without the anaphylactic side effects seen with traditional allergy vaccines.
↩[19] - Helm RM, Pomes A. Cockroach and other inhalant insect allergens. Clin Allergy Immunol 2004;18:271-296 ↩
[20] - Wang NM, Lee MF, Wu CH. Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen. Allergy 1999;54:119-127
BACKGROUND: Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties.
METHODS: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE.
RESULTS: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino- acid protein (50kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42.
CONCLUSIONS: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE- reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies
↩[21] - Wünschmann S, Gustchina A, Chapman MD, Pomés A. Cockroach allergen Bla g 2: An unusual aspartic proteinase. J Allergy Clin Immunol 2005;116:140-145
BACKGROUND: Enzymatic activity of mite, fungal, and bee venom allergens is thought to potentiate their allergenicity. Bla g 2 is a potent cockroach allergen, but despite sharing sequence homology with aspartic proteinases, it contains critical amino acid substitutions that impair proteolytic activity. The biologic function of Bla g 2 remains unclear .
OBJECTIVE: We sought to investigate the effects of specific amino acid substitutions on enzymatic activity, and the peptide-binding capability of Bla g 2 .
METHODS: Site-directed mutagenesis was used to produce a recombinant Bla g 2 mutant (Mut) with corrected canonical triads and a flap region. Another mutant (MutF - ) was expressed after additional mutations in the flap region of Mut. Bla g 2 wild-type (Wt), Mut, and MutF - were assayed for aspartic proteinase activity, and Bla g 2 Wt was tested for pepstatin binding .
RESULTS: Recombinant Bla g 2 Wt and Mut did not show enzymatic activity in a milk-clotting and hemoglobin assay. By using a modified hemoglobin assay, residual activity inhibited by pepstatin was detected for MutF - and Wt at 20 microg/mL, whereas pepsin was active at a 1000-fold lower concentration. Most of Bla g 2 binding to pepstatin-agarose was nonspecific .
CONCLUSION: Residual proteolytic activity was found for Bla g 2 at concentrations of approximately 4 mM. This weak activity suggests that proteolysis is not the primary function of this allergen and that it is unlikely to contribute to the allergenicity of Bla g 2. Bla g 2 has a cleft that might specifically bind ligands other than pepstatin.
↩[22] - Wünschmann S, Chapman MD, Gustchina A, Wlodawer A, Li M, Pomés A. Functional Study of the Cockroach Allergen Bla g 2. J Allergy Clin Immunol 2005;115(2 suppl.):S87
RATIONALE: Allergen proteolytic activity has been associated with IgE production and asthma. However, the major cockroach allergen Blag2, although homologous to aspartic proteinases, was inactive in standard aspartic proteinase assays. The absence of catalytic activity was attributed to critical amino acid changes in the catalytic site of the molecule Aim: To study the function and the potential for proteolytic activity of Blag2 by reverse engineering of the catalytic site
METHODS: Site-directed mutagenesis was used to introduce mutations in both catalytic triads (S33T, T34G, S217G) and the „flap‰ region (F75Y, F75a/deletion, or F75aA). Recombinant Blag2-wildtype (Wt) and mutants (Mut) were expressed in Pichia pastoris and affinity purified using 7C11 Mab. Blag2-Wt and Mut were tested for catalytic activity using a hemoglobin assay
RESULTS: Recombinant Blag2 (Wt and Mut) was inactive at concentrations of up to 80µg/ml when assayed in standard conditions, where pepsin is active at concentrations below 10µg/ml. However, using a modified assay with an extended incubation time of 16h, we detected residual activity not only for the mutant GTGK1 at 40µg/ml, but also for the Wt at 160µg/ml, whereas pepsin was already active at a 1000 fold lower concentration. Inhibition by pepstatin, but not PMSF (a serine protease inhibitor) indicated that the activity was aspartic proteinase
CONCLUSIONS: Residual proteolytic activity was found for Blag2 at concentrations of ~4mM. However, this very low activity suggests that proteolysis is not the primary function of this allergen and that it is unlikely to contribute to the allergenicity of Blag2
↩[23] - Pan QR, Wang SM, Shang HS, Chew FT. Identification and Characterization of Per a 2, the Bla g 2 Allergen Homologue from American Cockroach (Periplaneta americana). AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°450
RATIONALE: Cockroach hypersensitivity plays an increasingly important role in allergic diseases. We report the identification of an allergen (putatively named, Per a 2) from the american cockroach, Periplaneta americana, showing homology to Bla g 2
METHODS: We previously identified several expressed sequence tags showing sequence homology to Bla g 2. We multiple-aligned the sequences and then designed specific primers to obtain the full-length transcript. The coding regions of was cloned into a pET32 expression vector and recombinant histidine-tagged proteins expressed in Escherichia coli were obtained
RESULTS: The complete Per a 2 cDNA sequence is 1056bp long encoding 251 amino acids with a calculated molecular mass of approximately 38.1kDa. A predicted signal peptide with cleavage site at amino acid position 20-21 [VLC-DP] was observed. The translated protein sequence shared only 44% identity to Bla g 2, but had conserved aspartic acid and adjacent amino acids as with many aspartic proteases. However, the natural Per a 2 isolated by affinity chromatography did not show any aspartic proteinase activity when assayed via a milk clotting assay. Per a 2 however had a conserved Zn metal-binding site similar to that of Bla g 2, suggesting that it is possibly a Zn-binding inactivated aspartic protease. Recombinant Per a 2 was found to bind specific IgE in 28 of 53 cockroach allergic patients' sera ELISA inhibition studies show that Per a 2 and Bla g 2 shared low IgE cross-reactivity
CONCLUSIONS: We have thus identified the primary structure of an allergen, Per a 2, from the american cockroach Funding: Biomedical Research Council (BMRC) Singapore
↩[24] - Arruda LK. Cockroach allergens. Curr Allergy Asthma Rep 2005;5:411-416
Cockroaches produce several potent allergens associated with sensitization and the development of asthma. Many of these allergens have been cloned and produced as biologically active recombinant proteins. Three-dimensional structure and molecular modeling, in addition to studies on the biological functions of these proteins in the cockroach, have provided new insights into the ability of these allergens to cause immunoglobulin E antibody responses. Recent studies have pointed to a potential role of recombinant cockroach allergens for diagnosis and the development of novel therapeutic strategies that may benefit cockroach-allergic patients.
↩[25] - Pomés A, Chapman MD, Vailes LD, Blundell TL, Dhanaraj V. Cockroach Allergen Bla g 2. Structure, function, and implications for allergic sensitization. Am J Respir Crit Care Med 2002;165:391-397
Exposure to German cockroach (Blattella germanica) allergens is associated with the development of chronic respiratory diseases, especially asthma. The mechanism by which allergic patients develop specific immunoglobulin E (IgE) responses to environmental allergens is unknown. However, recent reports provided evidence that enzyme activity, especially proteolytic activity, was a major contributor to allergenicity. Bla g 2 is one of the most potent cockroach allergens (prevalence of IgE responses of 60 to 80%) and shows homology to the aspartic proteinase family of enzymes. We investigated whether the allergenicity of Bla g 2 was linked to its putative enzymatic function. A molecular model of Bla g 2, based on the high resolution crystal structures of pepsin and chymosin, showed that the overall three-dimensional structure of Bla g 2 was similar to that of aspartic proteinases with a well-defined binding pocket. However, critical amino acid substitutions in the catalytic triads and in the "flap" region of the molecule suggested that Bla g 2 was inactive and homologous to mammalian pregnancy-associated glycoproteins. This was confirmed experimentally by enzyme assay. The results show dissociation between enzymatic activity and allergenicity for Bla g 2 and suggest that other genetic and environmental factors are important determinants of sensitization.
↩[26] - Arruda LK, Vailes LD, Mann BJ, Shannon J, Fox JW, Vedvick TS, et al. Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases. J Biol Chem 1995;270:19563-19568
Inhalation of allergens produced by the German cockroach (Blattella germanica) elicits IgE antibody formation and the development of asthma in genetically predisposed individuals. We compared the allergenic importance of two cockroach (CR) allergens, Bla g 1 and Bla g 2, and determined the complete amino acid sequence of the major 36-kDa allergen, Bla g2. A survey of 106 sera from CR allergic patients showed the prevalence of IgE antibodies to Bla g 1 and Bla g 2 to be 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected patients gave positive reactions using 10(-3) micrograms/ml either allergen, whereas controls showed no response to 10 micrograms/ml. Natural Bla g 2 was purified and the sequence of the NH2 terminus and tryptic peptides, comprising 36% of the molecule, was determined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24-amino acid signal peptide and a 328-amino acid mature protein, which showed the highest degree of identity to mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%), with similar identity to pepsin, cathepsins D and E, renin, and chymosin. Bla g 2 mRNA and protein were detected in B. germanica, but not in Periplaneta americana, the other principal domiciliary CR species in the U.S. High concentrations of Bla g 2 were found in CR digestive organs (esophagus, gut, and proventriculus). The results show that Bla g 2 is a major species-specific allergen of B. germanica and suggest that the allergen functions as a digestive enzyme in the cockroach.
↩[27] - Wu HQ, Liu ZG, Gao B, Li M, Ran PX, Xing M. Localization of Per a 3 allergen in the gut and faecal pellets of the American cockroach (Periplaneta americana). Int J Immunogenet 2007;34:347-351
Cockroach-derived materials are known to be a major source of potent aeroallergens, causing allergic respiratory diseases such as asthma and allergic rhinitis. The aim of this study was to determine the localization of the major allergen, Per a 3 (Cr-PI), within the American cockroach (Periplaneta americana), which might reveal the relative importance of excreted materials and nonexcreted cockroach components as allergen sources. American cockroaches (P. americana) and their faecal pellets were embedded in paraffin, and serial sections were cut and collected on glass slides. After being stained with mouse polyclonal antiserum against Per a 3, the sections were incubated sequentially with biotin-labelled sheep antimouse immunoglobulin G (IgG) and a preformed fluorescent isothiocyanate (FITC)-avidin complex. Finally, the sections were mounted and examined under a fluorescent microscope. Examination of Per a 3 immunoreactivity on the sections of the American cockroaches (P. americana) revealed that the midgut mu cosa, gut contents and faecal pellets were all strongly labelled. Per a 3 immunoreactive products were not detected in any other internal organs of the American cockroaches. These results suggest that Per a 3 allergen might be synthesized in and secreted from the epithelia of the midgut mucosa and excreted from the body in the faecal pellets
↩[28] - Wu CH, Lee MF, Liao SC, Luo SF. Sequencing analysis of cDNA clones encoding the american cockroach Cr-PI allergens: homology with insect hemolymph proteins. J Biol Chem 1996;271:17937-17943
A previous article described the isolation of several lambdagt22A cDNA clones expressing the American cockroach (Periplaneta americana) Cr-PI allergens recognized by both human atopic IgE antibodies and anti-Cr-PI monoclonal antibodies (Wu, C. H., Lee, M. F., and Liao, S. C.(1995) J. Allergy Clin. Immunol. 96, 352-359). This article presents the nucleotide and deduced amino acid sequences of two cDNA clones encoding major allergens of P. americana. Clones C12 and C20 encode proteins of 685 and 631 amino acids with two potential N-glycosylation sites each. The predicted molecular weights for C12 and C20 cloned proteins are 79,300 and 75, 500 with isoelectric point values of 6.26 and 6.63, which are compatible with the determined sizes (Mr 78,000 and 72,000) and isoelectric point value (6.2) of the Cr-PI allergens of P. americana. A high degree of identity (69.1%), including several overlapped predicted central antigenic determinant residues, was found between two allergens. The anti-fusion protein antibody-based enzyme-linked immunosorbent assay was able to detect crude American cockroach extract, Cr-PI, recombinant proteins, and commercial cockroach extracts, which provides further evidence that two allergens share common antigen determinants. Recombinant allergens of clones C12 and C20 both showed 47.4% skin reactivities on 19 cockroach-sensitive asthmatic patients. Unexpectedly, although no sequence similarity was found to other known allergens, two aromatic amino acid-rich allergens were found to have a striking sequence identity to insect storage proteins (20.1-33.9%), insect juvenile hormone-suppressible proteins (30.9-36.4%), and arthropod hemocyanins (29.7-34.6%). Results suggested that two prominent allergens of P. americana are ancestrally related to these insect hemolymph proteins and represent a new group of proteins in the hemocyanin superfamily. These data will now facilitate epitope-mapping studies, and the recombinant allergens may be valuable for diagnostic and therapeutic purposes.
↩[29] - Bellinghausen I, Häringer B, Lafargue B, Strand D, König B, Decker H, et al. Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3. Clin Exp Allergy 2008;38:539-548
BACKGROUND: Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers .
OBJECTIVE: The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity .
METHODS: Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL-Per a 3), on proliferation and T-helper type 1 (Th1)/Th2 cytokine production of human CD4(+) T cells in co-culture with allergen-pulsed monocyte-derived autologous dendritic cells (DC) as well as the leukotriene release of basophils .
RESULTS: In P. americana-sensitized and non-sensitized donors the HL-Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN-gamma production than the hexamers. While in non-sensitized donors IL-4 and IL-5 as well as IL-10 production were also increased after stimulation with monomeric HL-Per a 3-pulsed DC, Th2 cytokine and IL-10 production were only enhanced in P. americana-sensitized donors using hexameric HL-Per a 3-pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers .
CONCLUSION: Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE-mediated allergic reaction and the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.
↩[30] - Wu CH, Lee MF, Wang NM, Luo SF. Sequencing and immunochemical characterization of the American cockroach Per a 3 (Cr-PI) isoallergenic variants. Mol Immunol 1997;34:1-8
Two additional members of the American cockroach (Periplaneta americana) Per a 3 (Cr-PI) allergen, C13 and C28, were isolated and sequenced. They encoded proteins of 470 and 393 amino acids with two and no potential N-glycosylation sites, respectively. The molecular weights for C13 and C28 cloned proteins are 56,200 and 46,7000, with PI values of 7.06 and 6.54. C13 and C28 display 95.4% identity with several overlapping predicted central antigenic determinants. Both allergens were also found to have a 95% sequence homology with previously cloned C20 and share similar antigenic determinants, as defined by the structural prediction and ELISA analysis. However, the recombinant C13 and C28 allergens showed 26.3 and 94.7% skin reactivities on asthmatic patients while C20 elicited 47.4%. While no sequence similarity was found to other known allergens, these two aromatic amino acid-rich allergens were highly related to insect hemolymph proteins (28.7-36.5%), as with C20 cloned protein. Results suggest that these two are isoallergenic variants of C20. Sequence variations among isoforms, resulting a significant difference in skin reactivities, will be useful in elucidating the allergenic determinants.
↩[31] - Arruda LK, Vailes LD, Hayden ML, Benjamin DC, Chapman MD. Cloning of cockroach allergen, Bla g 4, identifies ligand binding proteins (or calycins) as a cause of IgE antibody responses. J Biol Chem 1995;270:31196-31201
An allergen cloned from a Blattella germanica (German cockroach) cDNA library, encoded a 182-amino acid protein of 20,904 Da. This protein, designated B. germanica allergen 4 (Bla g 4), was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography and high-performance liquid chromatography. The prevalence of serum IgE antibody to recombinant Bla g 4 in 73 cockroach allergic patients with asthma ranged from 40% (antigen binding radioimmunoassay) to 60% (plaque immunoassay). Cockroach allergic patients gave positive intradermal skin tests to recombinant Bla g 4 at concentrations of 10(-3)-10(-5) micrograms/ml, whereas non-allergic controls, or cockroach allergic patients with no detectable serum IgE antibody to Bla g 4, gave negative skin tests to 1 microgram/ml. Polymerase chain reaction and Southern analysis identified a 523-base pair DNA encoding Bla g 4 in both B. germanica and Periplaneta americana (American cockroach). However, Northern analysis showed that mRNA encoding Bla g 4 was transcribed in B. germanica but not in P. americana, suggesting that allergen expression was species specific. Sequence similarity searches showed that Bla g 4 was a ligand binding protein or calycin and unexpectedly revealed that this family contained several important allergens: beta-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (approximately 20%), macromolecular modeling techniques were used to generate two models of the tertiary structure of Bla g 4, based on comparisons with the x-ray crystal coordinates of bilin binding protein and rodent urinary proteins. The results show that members of the calycin protein family can cause IgE antibody responses by inhalation or ingestion and are associated with asthma and food hypersensitivity.
↩[32] - Togias A, Poyser J, Satinover S, Collins D, Richards T, Green T, et al. IgE to Cockroach Allergens and Asthma. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°834
RATIONALE: Although allergy to cockroach has been strongly associated with asthma severity, only one study has found an association between cockroach allergy and asthma. Our goal was to examine whether such an association also exists within a homogeneous community that has ubiquitous exposure to cockroaches. Furthermore, we wanted to identify which cockroach allergens are the most common sensitizers
METHODS: We measured cockroach and dust mite serum IgE using CAP in 47 public housing residents from two Baltimore City communities We also measured specific IgE levels to various cockroach allergens Our subjects underwent extensive clinical evaluation including methacholine bronchoprovocation or albuterol reversibility testing for asthma phenotyping
RESULTS: Based on the clinical evaluation, we categorized the cohort in 3 groups: asthma (A, N=13), possible asthma (PA, N=11) and no asthma (NA, N=23). Cockroach IgE (IU/ml, mean ±SEM): 17.4±8.1 (A), 2.28±1.4 (PA), 3.0±2.0 (NA), p = 0.039. In contrast to cockroach, we found no difference between the 3 groups in D. pteronyssinus or D. farinae serum IgE. Positive IgE against Bla g 5 was the most common finding with Bla g 4, Blag 1, Bla g 2 and Per a 7 following in rank order
CONCLUSIONS: In Baltimore public housing residents, serum IgE against cockroach is a strong risk factor for asthma whereas IgE against dust mites is not. Bla g 5, a glutathione transferase protein, appears to be the major allergen in this population.
↩[33] - Santos AR, Rodrigues MC, Thiesen M, Zampolo AS, Ferriani VPL, Arruda L. Use of Recombinant Proteins for Diagnosis of Cockroach Allergy in Patients with Asthma and/or Rhinitis Living in Brazil. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°843
RATIONALE: Several cockroach allergens have been produced as recombinant proteins, however skin test reactivity to these allergens has been established in a limited number of patients
METHODS: Frequency of positive skin tests to recombinant allergens of Blattella germanica (rBlag 2, rBlag 4, rBlag 5) and Periplaneta americana (rPera 1 and rPera 7) was evaluated in 50 cockroach allergic patients with asthma and/or rhinitis, aged 7 to 70 years-old. Patients presented positive skin tests to P. americana and B. germanica. Skin prick tests with recombinant allergens were carried out at 10mcg/ml or 5mcg/ml (rPera 1) concentrations. Recombinant tropomyosins from P. americana (rPera 7) and A. lumbricoides, expressed in Pichia pastoris, were used in chimeric ELISA to quantitate IgE antibodies in sera of 48/50 patients
RESULTS: Positive skin tests to rBlag 2, rBlag 4, rBlag 5, rPera 1 and rPera 7 were found in 4(8%), 3(6%), 3(6%), 3(6%), and 18(36%) patients, respectively. Positive test to at least one allergen was found in 46% of patients. IgE antibodies to P. americana and A. lumbricoides tropomyosins were detected in 26(54.2%) and 25(52%) patients, respectively Levels of IgE to rPer a 7 varied from 0.6 to 200IU/mL (geometric mean GM 2.3IU/mL) and to A. lumbricoides tropomyosin, from 0.6 to 500IU/mL (GM 1.8IU/mL). There was a significant correlation of levels of IgE to Ascaris and P. americana tropomyosin (r=0.41; p=0.0034)
CONCLUSIONS: Recombinant allergens could be safely used for diagnosis of cockroach allergic patients living in Brazil. This approach could be improved by inclusion of additional cockroach allergens in the panel Funding:
↩[34] - Huang CH, Liew LM, Mah KW, Kuo IC, Lee BW, Chua KY. Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase. Clin Exp Allergy 2006;36:369-376
BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems .
OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST .
METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays .
RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively .
CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.
↩[35] - Jeong KY, Hong CS, Yong TS. Recombinant allergens for diagnosis and immunotherapy of allergic disorders, with emphasis on cockroach allergy. Curr Protein Pept Sci 2006;7:57-71
The prevalence of allergic disorders has increased over the past few decades and the quality of life has been significantly influenced at least for the allergic subjects. Allergen avoidance is thought to be the best way of preventing clinical manifestation of the disease, however, it is not possible for some allergens, and other pharmacological and/or immunological treatment has to be made. Repetitive injection of sensitized allergens to the patients (immunotherapy) is the only known curative approach to the disease even though the exact mechanism is not clear to date. Crude extract of allergens has lots of shortcomings which might arouse unexpected results. Genetic engineering and recombinant allergens are thought to be one of the alternative ways to overcome these limitations. Genetic engineering could facilitate the investigation of immune responses of the subjects especially on B cell and T cell epitopes, and produce the therapeutic allergens which might minimize the possible side effects. Furthermore, conjugation of immuno-modulatory molecules such as CpG-ODN, cytokines, or toxins which could act specifically to the given allergens, and maleylation of the allergens could maximize the prophylactic or therapeutic effect. Immunotherapies for the pollen allergy and insect sting allergy have been thought to be successful. House dust mite allergy and cockroach allergy have been reported less beneficial by immunotherapeutic approaches. Cockroaches are one of the most important causes of asthma, and severe complications are often reported in the children in city dwellers with low-incomes. The studies of the biological functions of cockroach allergens and the use of recombinant allergens should allow understanding of mechanisms of cockroach-elicited allergic disorders and development of allergen-specific and sensitive diagnostics and tailored therapeutic approaches in the future.
↩[36] - Hindley J, Wünschmann S, Satinover SM, Woodfolk JA, Chew FT, Chapman MD, et al. Bla g 6: A troponin C allergen from Blattella germanica with IgE binding calcium dependence. J Allergy Clin Immunol 2006;117:1389-1395
BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses .
OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy .
METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA .
RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens .
CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.
↩[37] - Hindley J, Wünschmann S, Satinover SM, Woodfolk JA, Chew FT, Chapman MD, et al. Bla g 6: A troponin C allergen from Blattella germanica with IgE binding calcium dependence. J Allergy Clin Immunol 2006;117:1389-1395
BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses .
OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy .
METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA .
RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens .
CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.
↩[38] - Asturias JA, Gomez-Bayon N, Arilla MC, Martinez A, Palacios R, Sanchez-Gascon F, et al. Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen. J Immunol 1999;162:4342-4348
Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved.
The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.
↩[39] - Crespo JF, Pascual CY, Helm R, Sanchez-Pastor S, Ojeda I, Romualdo L, et al. Cross-reactivity of IgE-binding components between boiled Atlantic shrimp and German cockroach. Allergy 1995;50:918-924
IgE-antibody reactivity to boiled Atlantic shrimp (Pandalus borealis) and German cockroach (Blattella germanica) of sera from 89 patients, sensitive to one or the other, was investigated with an enzymatic immunoassay for specific IgE detection (CAP-FEIA System, Pharmacia, Sweden). IgE serum levels to both antishrimp and anticockroach allergens were found to be positive in 76 of the 89 (85.4%) tested sera. A positive anticockroach IgE was very rare in the absence of detectable antishrimp IgE (five of 89 sera). Linear regression analysis on antishrimp and anti-German cockroach IgE levels-log plot revealed a correlation coefficient (r) of 0.73. Inhibition experiments showed that boiled Atlantic shrimp extract inhibited CAP with German cockroach, and vice versa. Immunoblotting showed the strongest IgE binding for both allergenic extracts between 30 and 43 kDa. By blot inhibition, the binding capacity of German cockroach was totally abolished by Atlantic shrimp extract, while German cockroach extract only partially IgE binding to Atlantic shrimp. Cross-reactivity exists between shrimp, an important food allergen, and German cockroach, which has an increasing role in allergic asthma. It could be important to determine the clinical significance of cross-allergy to both allergens, in which exposures occur in different ways.
↩[40] - Fernandes J, Reshef A, Patton L, Ayuso R, Reese G, Lehrer SB. IgE antibody reactivity to the major shrimp allergen, tropomyosin, in unexposed Orthodox Jews. Clin Exp Allergy 2003;33:956-961
BACKGROUND: Assessment of allergic (IgE antibody-mediated) reactions to foods may become complicated by cross-reactivity that can occur among certain food families and between foods and seemingly unrelated allergens .
OBJECTIVE: The allergenic properties of tropomyosin (muscle-derived protein) have been recently demonstrated in invertebrates such as cockroaches, dust mites, and shrimp. In view of a possible cross-reactivity between food allergens and related allergens from animal sources, we designed a study to assess IgE antibody reactivity to the major shrimp allergen, Pen a 1, in an unexposed population of Orthodox Jews, who observe Kosher dietary laws that prohibit eating shellfish .
METHODS: Nine subjects, who reacted positively by skin tests to shrimp (Penaeus setiferous), were selected for the study. Subjects (two females, seven males) ranged in age from 14 to 32 years (mean 20.4). All subjects were strictly observant of Jewish tradition and had no prior exposure to seafood (regarded as a non-Kosher food). Serum was obtained from all the subjects and tested for IgE antibody reactivity to shrimp and dust mite .
RESULTS: All subjects reported symptoms of perennial allergic rhinitis, five had history of asthma, atopic dermatitis, and/or sinusitis. All had positive skin prick tests to shrimp and house dust mite (HDM) (Dermatophagoides farinae, D. pteronyssinus, or both); 2/7 subjects were positive to cockroach mix (Blattella germanica and Periplaneta americana). Sera of 4/9 subjects demonstrated specific IgE antibodies by RAST to shrimp (7.0-20.0%), 3/9 to Pen a 1 (6.3-24.1%), and 3/9 to shrimp or Pen a 1 by immunoblot. IgE binding to Pen a 1 was inhibited with either mite or cockroach extracts as demonstrated by RAST and/or immunoblot inhibition analysis .
CONCLUSIONS: These studies indicate that IgE antibody reactivity to a major food allergen, shrimp, can occur in an unexposed population of individuals; some subjects allergic to HDM and/or cockroach show substantial IgE antibody reactivity to the major shrimp allergen Pen a 1 (tropomyosin). Based on inhibition with cockroach and/or dust mite extracts, this reactivity appears to be due to cross-reacting tropomyosins.
↩[41] - Riario-Sforza GG, Della Torre F, Antonicelli L, Bonifazi F, Giordano T, D'Amato G, et al. Sensitization to cockroach in Italy: a multicentric study. Allergy Asthma Proc 1997;18:23-28
The prevalence of sensitization to cockroach (CR) was evaluated by skin prick test (SPT) in 1299 patients consecutively referred for rhinitis and asthma to five allergy centers in northern, central, and southern Italy. In patients with positive SPT to CR, an SPT with D. pteronyssinus extract was made. RAST inhibition was performed with pooled sera of subjects with positive SPT and RAST of at least Class 3 for CR and D. Pteronyssinus, to assess the cross-reactivity between these two allergenic sources. The protein content of allergen extracts was assayed by isoelectric focusing (IEF). A total of 170 patients (13%) had a positive SPT with CR extract, and 133 of them (78.2%) were also positive for D. pteronyssinus. RAST inhibition showed a cross-reactivity between CR and D. pteronyssinus, and IEF detected in the extracts employed a series of bands focusing at the same pI of CR and D. pteronyssinus relevant allergens. Therefore, the prevalence of sensitization to CR in the population studied, corresponding to 13%, suggests that CR extract might be added to standard SPT panels for respiratory allergy. However, the cross-reactivity with D. pteronyssinus calls for careful evaluation of the clinical importance of this sensitization.
↩[42] - Jeong KY, Hwang H, Lee J, Lee IY, Kim DS, Hong CS, et al. Allergenic characterization of tropomyosin from the dusky brown cockroach, Periplaneta fuliginosa. Clin Diagn Lab Immunol 2004;11:680-685
Household arthropods are one of the most common causes of allergic diseases. Four species of cockroaches are found to reside in Korean homes, but published work deals almost exclusively with the German and American cockroaches. This study was undertaken to investigate the cross-reactive allergenic components of the dusky brown cockroach, Periplaneta fuliginosa. Enzyme-linked immunosorbent assay (ELISA) inhibition and immunoblot analyses for the dusky brown cockroach were performed with Blattella germanica and Dermatophagoides farinae allergic sera. cDNA encoding tropomyosin, which is a well known cross-reactive pan-allergen, was cloned by reverse transcriptase PCR, and recombinant protein was produced by using a pET-28b expression system. Native tropomyosin was purified by ammonium sulfate fractionation and electroelution. The immunoglobulin E (IgE) reactivities of native and recombinant tropomyosins were compared by an ELISA inhibition study. All 30 sera tested showed P. fuliginosa-specific IgE, and the IgE-binding reactivity of the P. fuliginosa extract was inhibited as much as 79.4% by a B. germanica extract and as much as 63.3% by a D. farinae extract. The deduced amino acid sequence of cloned cDNA was identical with that of Periplaneta americana tropomyosin (98.5% nucleotide sequence identity). Seven of 26 (26.9%) allergic sera had IgE specific for recombinant protein, and the maximum inhibition of P. fuliginosa-specific IgE achieved with recombinant tropomyosin was 37.7% at an inhibitor concentration of 10 microg/ml. Native tropomyosin inhibited the binding of IgE to the P. fuliginosa, B. germanica, and D. farinae extracts by 65.0, 51.8, and 39% at an inhibitor concentration of 1 microg/ml. P. fuliginosa appears to possess allergens that are highly cross-reactive with allergens of B. germanica and D. farinae. Tropomyosin was found to be a major allergenic component accounting for the cross-reactivity between cockroaches and dust mites.
↩[43] - Witteman AM, van den Oudenrijn S, van Leeuwen J, Akkerdaas J, van der Zee JS, Aalberse RC. IgE antibodies reactive with silverfish, cockroach and chironomid are frequently found in mite-positive allergic patients. Int Arch Allergy Immunol 1995;108:165-169
Approximately 30% of the house dust mite allergic patients in The Netherlands have IgE antibodies reactive with silverfish, cockroach and/or chironomid. In allergic patients without IgE antibodies against Dermatophagoides pteronyssinus less than 5% have IgE antibodies reactive with these insects. By means of RAST inhibition studies it is shown that cross-reactivity exists between D. pteronyssinus and silverfish, cockroach or chironomid. This means that a positive RAST for silverfish, cockroach, chironomid or D. pteronyssinus cannot be taken as evidence for exposure.
↩[44] - Jeong KY, Hwang H, Lee J, Lee IY, Kim DS, Hong CS, et al. Allergenic characterization of tropomyosin from the dusky brown cockroach, Periplaneta fuliginosa. Clin Diagn Lab Immunol 2004;11:680-685
Household arthropods are one of the most common causes of allergic diseases. Four species of cockroaches are found to reside in Korean homes, but published work deals almost exclusively with the German and American cockroaches. This study was undertaken to investigate the cross-reactive allergenic components of the dusky brown cockroach, Periplaneta fuliginosa. Enzyme-linked immunosorbent assay (ELISA) inhibition and immunoblot analyses for the dusky brown cockroach were performed with Blattella germanica and Dermatophagoides farinae allergic sera. cDNA encoding tropomyosin, which is a well known cross-reactive pan-allergen, was cloned by reverse transcriptase PCR, and recombinant protein was produced by using a pET-28b expression system. Native tropomyosin was purified by ammonium sulfate fractionation and electroelution. The immunoglobulin E (IgE) reactivities of native and recombinant tropomyosins were compared by an ELISA inhibition study. All 30 sera tested showed P. fuliginosa-specific IgE, and the IgE-binding reactivity of the P. fuliginosa extract was inhibited as much as 79.4% by a B. germanica extract and as much as 63.3% by a D. farinae extract. The deduced amino acid sequence of cloned cDNA was identical with that of Periplaneta americana tropomyosin (98.5% nucleotide sequence identity). Seven of 26 (26.9%) allergic sera had IgE specific for recombinant protein, and the maximum inhibition of P. fuliginosa-specific IgE achieved with recombinant tropomyosin was 37.7% at an inhibitor concentration of 10 microg/ml. Native tropomyosin inhibited the binding of IgE to the P. fuliginosa, B. germanica, and D. farinae extracts by 65.0, 51.8, and 39% at an inhibitor concentration of 1 microg/ml. P. fuliginosa appears to possess allergens that are highly cross-reactive with allergens of B. germanica and D. farinae. Tropomyosin was found to be a major allergenic component accounting for the cross-reactivity between cockroaches and dust mites.
↩[45] - Arruda LK. Cockroach allergens. Curr Allergy Asthma Rep 2005;5:411-416
Cockroaches produce several potent allergens associated with sensitization and the development of asthma. Many of these allergens have been cloned and produced as biologically active recombinant proteins. Three-dimensional structure and molecular modeling, in addition to studies on the biological functions of these proteins in the cockroach, have provided new insights into the ability of these allergens to cause immunoglobulin E antibody responses. Recent studies have pointed to a potential role of recombinant cockroach allergens for diagnosis and the development of novel therapeutic strategies that may benefit cockroach-allergic patients.
↩[46] - Ayuso R, Grishina G, Bardina L, Carrillo T, Blanco C, Ibañez MD, et al. Myosin light chain is a novel shrimp allergen, Lit v 3. J Allergy Clin Immunol 2008;122:795-802
BACKGROUND: Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. Few options are available for treatment, and avoidance is the only therapy recommended .
OBJECTIVE: We sought to identify relevant crustacean allergens for use as diagnostic and safe immunotherapeutic agents for subjects with shellfish allergy .
METHODS: Thirty-eight patients were selected with immediate allergic reactions to shrimp and increased shrimp-specific serum IgE levels. One-dimensional and 2-dimensional electrophoresis of shrimp extracts were followed by IgE immunoblotting. Protein identification was done with matrix-assisted laser desorption/ionization-mass spectrometry and Edman sequencing. A cDNA library was generated from white pacific shrimp (Litopenaeus vannamei) and screened with primers designed on the basis of internal sequences obtained from 2-dimensional tryptic digests. Full-length cDNA clones were isolated from the library and sequenced. Recombinant protein was expressed and tested with sera from patients with shrimp allergy .
RESULTS: Immunoblotting demonstrated IgE binding to a 20-kDa shrimp protein by 21 (55%) of 38 sera. Tryptic digestion of the protein followed by matrix-assisted laser desorption/ionization-mass spectrometric analysis and Edman sequencing identified it as a myosin light chain (MLC). Screening of the shrimp cDNA library resulted in isolation of a novel protein cDNA. Open reading frame translation provided the amino acid sequence of a new allergenic shrimp protein with high similarity to Bla g 8 (cockroach MLC). Recombinant protein was recognized by 17 patients, confirming the allergenicity of shrimp MLC .
CONCLUSIONS: We have identified and cloned a new major shrimp allergen, Lit v 3.0101, an MLC protein.
↩[47] - Lafargue B, Tenzer S, Decker H. Biophysical characterization of arginin-kinase, a new pan-allergen of the American cockroach. Allergy 2007;62(suppl. 83):302
Background Allergy against cockroach is a worldwide problem. It was clearly demonstrated that asthma caused by cockroaches is antigen specific and similar to other types of atopic asthma.The incidence of asthmatic patients who are sensitized to cockroach allergens ranges from 40% to 70% depending on the geographical location. The most common cockroaches are the German cockroach and the American cockroach. So far, several allergens of the American cockroach are characterized, but only one study was engaged in the allergenicity of the arginine kinase: the native arginine kinase reacted to IgE in 80% and 100% of cockroach-allergic Thai patients. Homologous proteins, thus most likely being allergens as well, were found in many species. Thus, the arginine kinase is not only a major allergen of the American cockroach but shall also be discussed as a new pan-allergen.
Method Arginine kinase was purified of an American cockroach crude-extract in a two step protocol employing anion-exchange chromatography and size exclusion chromatography. The molecular mass of the purified enzyme was analyzed with SDS-PAGE and an analytical ultracentrifuge equilibrium run. Further characterization was determined with analytical ultracentrifuge sedimentation run, dynamic light scattering and absorption spectra. The purified protein was identified by LC-MSMS analysis of a tryptic digest of the most prominent SDS-PAGE gel band.
Results We were able to isolate 6mg arginine kinase of 18ml American cockroach crude extract. The isolated protein was identified by ESI-QTOF mass spectrometry as arginin-kinase (AAT77152) and was only detected in the crude-extract and not in the hemolymph of the cockroach. The molecular mass of the native protein was determined by analytical ultracentrifugation to about 33 kDa and of the denaturated enzyme to 39 kDa. No difference between the reduced and non-reduced enzyme was observed on SDS-PAGE. All these data fit well with the molecular dimensions of 48-56 obtained with dynamic light scattering and the corrected sedimentation coefficient of 3S. In addition to the typical aromatic amino acid peak at 280nm, the absorption spectra showed a weak absorption at 408 nm. Conclusions We have clearly demonstrated that the arginine kinase of the American cockroach occurs only in a cockroach crude-extract and not in the hemolymph. Arginine kinase is a spherical monomeric allergen with no inter- or intramolecular disulfide-bridges.
↩[48] - Panzani RC, Ariano R. Arthropods and invertebrates allergy (with the exclusion of mites): the concept of panallergy. Allergy 2001;56(suppl. 69):1-22 ↩
[49] - Ock MS, Kim BJ, Kim SM, Byun KH. Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen. Korean J Parasitol 2005;43:101-110
In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited 42-57% homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GDSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cockroach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.
↩[50] - Sudha VT, Arora N, Gaur SN, Pasha S, Singh BP. Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana. Allergy 2008;63:768-776
BACKGROUND: Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity .
METHODS: A serine protease was isolated from P. americana extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation .
RESULTS: Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with mite serine protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine protease inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency .
CONCLUSION: A serine protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of allergy and will be useful in elucidating the mechanism of allergy.
↩[51] - Ma B, Chang FN. Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica. FEBS J 2007;274:1793-1803
A highly active glutathione S-transferase was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24 kDa, and had a different electrophoretic mobility than glutathione S-transferase (Bla g 5). This glutathione S-transferase may play a role in defense against insecticides as well as oxidative stress. The sequence had close similarities (approximately 45-60%) to that of Delta class glutathione S-transferases, but had only 14% identity to Bla g 5. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.
↩[52] - Jeong KY, Jeong KJ, Yi MH, Lee H, Hong CS, Yong TS. Allergenicity of Sigma and Delta Class Glutathione S-Transferases from the German Cockroach. Int Arch Allergy Immunol 2009;148:59-64
BACKGROUND: Cockroach glutathione S-transferases (GSTs) are known to elicit strong IgE responses. This study was undertaken to compare the IgE reactivity of German cockroach GSTs, Bla g 5 (sigma class) and delta class GST (BgGSTD1) .
METHODS: Full-length Bla g 5 and BgGSTD1 were cloned, and their recombinant proteins were expressed and purified. Their IgE reactivities and cross-reactivities were examined by ELISA using sera from cockroach-sensitized subjects .
RESULTS: A predominant variant of Bla g 5 cDNA has amino acid substitutions at positions 10 (C to F) and 42 (N to K). BgGSTD1 has substitutions at positions 27 (E to N) and 207 (K to R). Sera from cockroach-sensitized patients showed 20.5% IgE reactivity to Bla g 5 and 17.9% IgE reactivity to BgGSTD1. However, inhibition studies using 1 serum sample with the highest IgE reactivity showed limited cross-reactivity .
CONCLUSIONS: IgE-binding frequency to the cockroach GSTs was low, but the titer of IgE reactivity was strong in some sera. The inclusion of different classes of GSTs could be helpful for the delicate diagnosis and immunotherapy of cockroach allergy.
↩[53] - Khantisitthiporn O, Sookrung N, Tungtrongchitr A, Tongtawe P, Bunnag C, Srimanote P, et al. Native troponin-T of the American cockroach (CR), Periplaneta americana, binds to IgE in sera of CR allergic Thais. Asian Pac J Allergy Immunol 2007;25:189-197
The American cockroach, Periplaneta americana, is the predominant cockroach (CR) species in Thailand and a major source of indoor allergens second only to the house dust mite. The incidence of CR allergy among allergic Thai patients is increasing but basic information on the allergenic components is scarce. In this study a recombinant troponin-T was produced by using cDNA prepared from RNA of the P. americana as a template and PCR primers designed from the P. americana troponin-T sequence deposited in the GenBank database. The recombinant protein (Mr approximately 50) did not bind to IgE in the sera of 18 skin prick test positive CR allergic patients. Rabbit polyclonal antiserum (PAb) against the recombinant troponin-T was produced and used in preparing an affinity column for the purification of native troponin-T from the crude P. americana extract (Mr approximately 47). IgE-immunoblotting revealed that the native protein bound to IgE in 3 of the 18 (16.7%) patients. Our results imply that native P. americana troponin-T, but not its recombinant counterpart, is a minor allergen among the CR allergic Thais.
↩[54] - Duong PT, Chang FN. A simple method for assigning multiple immunogens to their protein on a two-dimensional blot and its application to asthma-causing allergens. Electrophoresis 2001;22:2098-2102
A "one-step" procedure, that not only removes the color and blocking proteins used in the colorimetric immunodetection step but also stains the proteins originally on the blot, is presented. Following immunostaining and recording of immunoreactive spots, the blot was allowed to air-dry overnight (or longer) at room temperature and then counterstained with a colloidal gold solution. This "air-drying" process apparently altered the affinity of the blocking proteins (and possibly other proteins added subsequently to the blotting step) towards the nitrocellulose membrane causing them to be removed by the acidic colloidal gold solution while the "blotted" proteins were being stained. The sensitivity of this counterstained blot was comparable to that of the blot without going through the immunodetection process. Since both immunodetection and protein staining were carried out on the same blot, this allowed easy identification of many immunoreactive spots to their corresponding proteins when the two profiles were superimposed. Using this procedure, we have detected 25 immunoreactive spots (or allergens) from the whole body extract of the German cockroach (Blattella germanica) that contribute to asthma and assigned them to their corresponding proteins on a two-dimensional (2-D) protein map. The apparent Mr and pI for each of the allergens were determined. We have also located one of the major cockroach allergens, Bla g 5 (glutathione S-transferase). Two-dimensional zymography revealed the presence of ten gelatinase-type proteolytic enzymes. Only one of the ten proteases comigrated with the immunoreactive proteins indicating that unlike other allergen-producing systems, most of the cockroach allergens do not possess protease activity.
↩[55] - Wünschmann S, Satinover SM, Woodfolk JA, Chapman MD, Pomés A. Purification and Characterization of Blattella germanica Serine Protease. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°453
RATIONALE: Many aeroallergens possess proteolytic activity, including dust mite serine proteases Der p 3, Der p 6, and Der p 9. Serine proteases in Blattella germanica (B.g.) extract have been associated with inflammation of human airway epithelial cells. However little is known about their allergenicity. We investigated IgE reactivity to serine protease purified from B.g. extract
METHODS: Serine protease was purified from B.g. frass by affinity chromatography using immobilized Soybean Trypsin Inhibitor (STI) and was analyzed by silver-stained SDS PAGE. A milk clotting assay was used to assess enzymatic activity and IgE reactivity was analyzed by immunoblot and a multiplex fluorescent assay. The multiplex assay used serine protease coupled to beads to measure IgE binding from sera of highly cockroach allergic patients (n = 15; 9/15 sera had IgE levels ranging from 10 IU/ml - 88.3 IU/ml to cockroach extract)
RESULTS: SDS-PAGE of STI-affinity purified serine protease from B.g identified a ~25kD protein, which was proteolytically active in a milk clotting assay at less than 9.37µg/ml. Enzymatic activity was inhibited by the specific serine protease inhibitor phenylmethylsulfonyl fluoride. None of the 15 sera tested showed significant IgE reactivity to the B.g. serine protease in immunoblot or multiplex assay
CONCLUSIONS: The Blattella germanica serine protease, although proteolytically active, does not appear to be allergenic in the highly cockroach allergic patients tested. Active cockroach serine protease may have direct effects on the airways without inducing IgE mediated sensitization Funding: INDOOR Biotechnologies Inc.
↩[56] - Wada K, Matsuwaki Y, Bingemann TA, Kita H. Cockroach Activates and Induces Degranulation of Human Eosinophils Through Protease-Activated Receptor-2. J Allergy Clin Immunol 2007;119(1 suppl):S215
RATIONALE: Exposure to cockroach is a major risk factor for bronchial asthma, and the enzymatic activities of cockroach allergens are implicated in their allergenicity. Because little is known about the immunological properties of cockroach, we investigated the effects of cockroach on eosinophil activation in vitro.
METHODS: Eosinophils were isolated from normal individuals or patients with mild allergy or asthma and incubated with extracts of German, Oriental or American cockroaches for up to 3 hours. Activation of eosinophils was monitored by degranulation and superoxide anion production. Enzymatic activities of cockroach extracts were studied by using fluorescence substrates.
RESULTS: German and Oriental cockroach extracts, but not American cockroach extract, induced superoxide anion production and eosinophil degranulation in a concentration-dependent manner. The rank order of potency was German>Oriental>American. Eosinophil degranulation was observed in eosinophils from both normal individuals and patients. German and Oriental cockroach extracts cleaved a malaria aspartate protease substrate and a specific substrate mimicking the N-terminus (33-43) of protease-activated receptor (PAR)-2. These enzymatic activities of German and Oriental cockroach extracts were inhibited by heat-treatment at 56 °C for 30 minutes or affinity chromatography with pepstatin agarose (an aspartate protease inhibitor). Finally, eosinophil degranulation induced by German cockroach extract was inhibited by preincubation of eosinophils with a specific PAR-2 inhibitory peptide or by pretreatment of the extract with pepstatin agarose.
CONCLUSIONS: Certain cockroaches produce immunoactive molecules, likely aspartate protease(s), that activate eosinophils -even from normal individuals- through PAR-2 and induce degranulation. The immunological activities of cockroach molecules may be involved in airway inflammation and asthma exacerbation.
↩[57] - Page K, Hughes VS, Bennett GW, Wong HR. German cockroach proteases regulate matrix metalloproteinase-9 in human bronchial epithelial cells. Allergy 2006;61:988-995
BACKGROUND: Matrix metalloproteinases (MMPs) digest extracellular matrix proteins and may play a role in the pathogenesis of bronchial asthma. MMP-9 levels are increased in the bronchoalveolar lavage fluid and sputum of asthmatics compared with that of controls. As exposure to cockroaches is an environmental risk factor for asthma, we sought to investigate the role of German cockroach fecal remnants (frass) on MMP-9 expression .
METHODS: Human bronchial epithelial cells (16HBE14o-) and primary normal human bronchial epithelial cells were treated with cockroach frass in the absence or presence of tumor necrosis factor (TNF)alpha. MMP-9 mRNA, protein levels and pro-MMP-9 activity were determined using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and zymogram assays. Pretreatment of frass with aprotinin abolished protease activity. PD98059, a chemical inhibitor of extracellular signal regulated kinase (ERK), and SLIGKV, an activator of protease-activated receptor (PAR)-2 were also used. AP-1DNA binding was determined by electrophoretic mobility shift assay (EMSA) and ERK phosphorylation by Western blot analysis .
RESULTS: Cockroach frass augmented TNFalpha-mediated MMP-9 mRNA and protein expression by a mechanism dependent on active serine proteases within frass and not on endogenous endotoxin. Frass increased ERK phosphorylation, and chemical inhibition of ERK attenuated cockroaches' effects on MMP-9. Serine proteases are known to activate the PAR-2 receptor. We found that selective activation of PAR-2 using the peptide SLIGKV augmented TNFalpha-induced MMP-9 protein levels and increased ERK phosphorylation. Frass and SLIGKV each increased AP-1 translocation and DNA binding .
CONCLUSIONS: These data suggest that German cockroach frass contains active serine proteases which augment TNFalpha-induced MMP-9 expression by a mechanism involving PAR-2, ERK and AP-1.
↩[58] - Hong JH, Lee SI, Kim KE, Yong TS, Sohn MH, Shin DM. German cockroach extract activates protease-activated receptor 2 in human airway epithelial cells. J Allergy Clin Immunol 2004;113:315-319
Background The German cockroach has been reported to act as an allergen that might be associated with a protease reaction in asthma. However, the molecular identities of the antigens in German cockroach extract (GCE) with protease activity and the protease-activated receptors (PARs) that are activated by GCE in human airway epithelial cells have not been characterized. Objective : We investigated the direct effect of GCE on Ca2+ signaling in human airway epithelial cells and the type of PARs activated by GCE. Method s : The Ca2+-sensitive dye Fura2 was used to determine intracellular Ca2+ concentration ([Ca2+]i) by means of spectrofluorometry. Result s : GCE induced a baseline type of [Ca2+]i oscillations in a dose-dependent manner. The oscillations persisted for long periods of time in the absence of Ca2+ entry across the plasma membrane, suggesting that the observed [Ca2+]i increases were due to Ca2+ release from intracellular stores. Accordingly, after depleting endoplasmic reticulum Ca2+ with thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, the GCE-mediated [Ca2+]i signals were abolished. Whereas desensitization of PAR-1, PAR-3, and PAR-4 had no effect on GCE-mediated Ca2+ mobilization, no GCE-mediated [Ca2+]i increase was observed after desensitization of PAR-2. Conclusions : These results indicate that GCE has a direct effect on human airway epithelial cells, in particular generating [Ca2+]i oscillations through Ca2+ release from thapsigargin-sensitive Ca2+ stores through activation of PAR-2.
↩[59] - Sohn MH, Lee YA, Jeong KY, Sim S, Kim KE, Yong TS, et al. German Cockroach Extract Induces Activation of Human Eosinophils to Release Cytotoxic Inflammatory Mediators. Int Arch Allergy Immunol 2004;134:141-149
BACKGROUND: Eosinophils play an important role in the pathogenesis of allergic diseases. Sensitization and exposure to cockroach allergen have been demonstrated to be one of the major risk factors for the development of bronchial asthma. However, little is known regarding the functional capacity of cockroach extract antigen to activate human eosinophils .
OBJECTIVE: We investigated whether German cockroach extract can activate human eosinophils to release cytotoxic inflammatory mediators .
METHODS: Purified eosinophils from the peripheral blood were incubated with various concentrations (0-200 microg/ml) of German cockroach extract antigen. Effector functions of eosinophils were checked by degranulation and superoxide anion production. In addition, we examined surface expression of CD11b and CD69, and intracellular activation of p38 mitogen-activated protein kinase (p38 MAP kinase) in cockroach-stimulated eosinophils .
RESULTS: German cockroach extract induced degranulation and superoxide production from human eosinophils. In addition, incubation of eosinophils for 3 h with the cockroach extract resulted in an increased level of the surface expression of CD11b and CD69. Furthermore, cockroach-induced superoxide production from eosinophils was significantly inhibited by the pretreatment of cells with a p38 MAP kinase inhibitor SB202190. Indeed, a large amount of phosphorylated forms of p38 MAP kinase was detected in cockroach-stimulated eosinophils .
CONCLUSIONS: Our results suggest that German cockroach extract induces activation of human eosinophils to release cytotoxic inflammatory mediators such as superoxide and granular proteins.
↩[60] - Bhat RK, Page K, Tan A, Hershenson MB. German cockroach extract increases bronchial epithelial cell interleukin-8 expression. Clin Exp Allergy 2003;33:35-42
BACKGROUND: Cockroach exposure has been recognized as a common trigger for asthma. While dust mite and Aspergillus fumigatus aeroallergens have been noted to have direct effects on airway epithelium, direct effects of cockroach proteins have not been determined .
OBJECTIVE: The purpose of this study was to investigate whether cockroach extract has a direct pro-inflammatory effect on airway epithelium .
METHODS: We examined the effect of crude German cockroach (Blattella germanica) extract on IL-8 expression in a human bronchial epithelial cell line (16HBE14o-cells) and primary human bronchial epithelial cells. Transcription from the IL-8 promoter and protein abundance were determined by reporter assay and enzyme-linked immunosorbent assay (ELISA), respectively. Endotoxin levels in the crude cockroach extracts were determined using the Limulus Amebocyte Lysate assay. Protease activity was assessed using Azocoll as a substrate .
RESULTS: We found that crude cockroach extract induced a synergistic increase in TNF-alpha-induced transcription from the IL-8 promoter. The synergistic effect was observed with as little as 0.3 micro g/mL of crude cockroach extract, while larger concentrations (30 micro g/mL) approximately doubled TNF-alpha-induced IL-8 promoter activity. Similar effects of cockroach extract on IL-8 protein abundance were observed in both 16HBE14o- and primary human bronchial epithelial cells. Addition of endotoxin at concentrations found in the cockroach extract had no effect on TNF-alpha-mediated IL-8 expression. The serine protease inhibitors aprotinin and phenylmethylsulphonyl fluoride abolished cockroach-induced synergy, while the cysteine protease inhibitors E64 and leupeptin had little effect. Measurement of protease activity using Azocoll as a substrate confirmed the presence of protease activity in cockroach extracts. Addition of recombinant Bla g 2, Bla g 3 and Bla g 5 had no effect on TNF-alpha-induced IL-8 promoter activation. Finally, cockroach extract also increased TNF-alpha-induced transcription from the IL-6 promoter .
CONCLUSIONS: German cockroach extract contains novel serine protease activity which has a direct pro-inflammatory effect on airway epithelial cells
↩[61] - Page K, Strunk VS, Hershenson MB. Cockroach proteases increase IL-8 expression in human bronchial epithelial cells via activation of protease-activated receptor (PAR)–2 and extracellular-signalregulated kinase. J Allergy Clin Immunol 2003;112:1112-1118
BACKGROUND: We have shown that serine proteases in German cockroach extract increase TNF-alpha-induced expression of IL-8 in human bronchial epithelial cells. The mechanism by which cockroach proteases regulate cytokine expression is unknown; however, protease-activated receptors (PARs) might play a role .
OBJECTIVE: We sought to determine the role of PARs and extracellular-signal-regulated kinase (ERK) in cockroach-induced regulation of IL-8 expression .
METHODS: 16HBE14o- human bronchial epithelial cells were treated with the specific PAR-1 and PAR-2 agonists, TFRIFD and SLIGKV, respectively. IL-8 transcription was assessed by transiently transfecting cells with a luciferase-tagged IL-8 promoter construct, and in some cases, dominant-negative expression vectors. To block PAR cleavage, antibodies against the cleavage region of PAR-1 and PAR-2 were used. ERK phosphorylation was determined by Western blot .
RESULTS: Although both PAR-1 and PAR-2 were endogenously expressed in 16HBE14o- cells, selective activation of PAR-2 but not PAR-1 mimicked the effect of cockroach extract on IL-8 expression. Using a blocking antibody against cleavage of PAR-2 but not PAR-1 attenuated cockroach-extract-induced responses, suggesting that cockroach proteases cleave PAR-2. Treatment of cells with cockroach extract and SLIGKV each increased phosphorylation of ERK. Chemical or genetic inhibition of Ras and mitogen-activated protein kinase/ERK (MEK), upstream activators of ERK, each attenuated cockroach- and PAR-2-induced IL-8 transcription .
CONCLUSION: Cockroach proteases and PAR-2 activation synergistically increase TNF-alpha-induced IL-8 transcription via activation of ERK. These data suggest an important role for PAR-2 and ERK activation in the regulation of cytokine expression in airway epithelium in response to cockroach proteases.
↩[62] - Kuderer NM, San-Juan-Vergara HG, Kong X, Esch R, Lockey RF, Mohapatra SS. Mite and cockroach proteases activate p44/p42 MAP kinases in human lung epithelial cells. Clin Mol Allergy 2003;1:1
BACKGROUND: The mechanisms underlying epithelial cell activation by indoor inhaled antigens are poorly understood .
METHODS: In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 epithelial cells upon exposure to antigens of house dust mite (HDMA), German cockroach (GCA), and American cockroach (ACA) .
RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control. Exposure of A549 cells to these antigens induced the phosphorylation of p44/42 MAPKs within 5 minutes, which reached a peak at 25 minutes later and reached baseline levels at 1 hour after exposure. PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production. Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production .
CONCLUSION: Thus, proteolytic antigens present in HDMA, GCA and ACA activate the p44/42 MAPKs airway epithelial cells, which lead to elevated IL-8 production and initiation of the inflammatory cascade.
↩[63] - Antony AB, Tepper RS, Mohammed KA. Cockroach extract antigen increases bronchial airway epithelial permeability. J Allergy Clin Immunol 2002;110:589-595
BACKGROUND: The bronchial epithelial cells of airways are subject to recurrent environmental injury throughout the life of an individual. Recently, a high incidence of asthma has been reported in inner-city children. The increased incidence of asthma in inner-city children is thought to be caused, in part, by frequent exposure to allergens of the common household pest the cockroach .
OBJECTIVE: We sought to investigate whether cockroach extract antigen (CrAg) induces vascular permeability factor, also known as vascular endothelial growth factor (VEGF), and whether it increases permeability in bronchial airway epithelial cells (BAECs) .
METHODS: We estimated CrAg-induced VEGF release in BAECs by using an ELISA and VEGF mRNA expression by using an RT-PCR reaction. The influence of CrAg on BAEC barrier function was estimated by measuring electrical resistance with an electric cell substrate impedance-sensing system .
RESULTS: Our results demonstrate that CrAg induces VEGF release in BAECs in a time-dependent manner. The VEGF induction was also confirmed by means of VEGF mRNA expression in CrAg-stimulated BAECs. CrAg decreased electrical resistance across BAEC monolayers. The maximum decrease in electrical resistance was noticed 6 hours after activation and reached a plateau thereafter. Neutralizing antibodies to VEGF significantly inhibited the decrease in BAEC electrical resistance caused by CrAg .
CONCLUSIONS: These data suggest that CrAg induces VEGF release in BAECs and alters bronchial airway permeability.
↩[64] - Hughes VS, Page K. German cockroach frass proteases cleave pro-matrix metalloproteinase-9. Exp Lung Res 2007;33:135-140
Matrix metalloproteinase (MMP)-9, secreted as pro-MMP-9, is cleaved by serine proteases at the N-terminus to generate active MMP-9. Pro-MMP-9 has been found in the bronchoalveolar lavage fluid of patients with asthma. Because many inhaled aeroallergens contain active proteases, the authors sought to determine whether German cockroach (GC) fecal remnants (frass) and house dust mite (HDM) were able to cleave pro-MMP-9. Treatment of recombinant human (rh) pro-MMP-9 with GC frass resulted in a dose- and time-dependent cleavage. This was abrogated by pretreating frass with an inhibitor of serine, but not cysteine protease activity. GC frass also induced cleavage of pro-MMP-9 from primary human neutrophils dependent on the active serine proteases in GC frass. HDM was less potent at cleaving pro-MMP-9. Alpha1-antitrypsin (A1AT), a naturally occurring protease inhibitor, attenuated GC frass-induced cleavage of pro-MMP-9. A1AT partially inactivated the serine protease activity in GC frass, while GC frass cleaved A1AT in a dose- and time-dependent manner. These data suggest that GC frass-derived serine proteases could regulate the activity of MMP-9 and that A1AT may play an important role in modulating GC frass activity in vivo. These data suggest a mechanism by which inhalation of GC frass could regulate airway remodeling through the activation of pro-MMP-9.
↩[65] - Panzani RC, Ariano R. Arthropods and invertebrates allergy (with the exclusion of mites): the concept of panallergy. Allergy 2001;56(suppl. 69):1-22 ↩
[66] - Satinover SM, Reefer AJ, Pomes A, Chapman MD, Platts-Mills TAE, Woodfolk JA. Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 2005;115:803-809
BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied .
OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects .
METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques .
RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon .
CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.
↩[67] - Sakaguchi M, Inouye S, Miyazawa H, Okabe T, Yasueda H, Muto A, et al. Sensitization to cockroach allergens of asthma patients in Japan. Arerugi 1994;43:1309-1315
To evaluate the role of allergens from Periplaneta fuliginosa, which is the most predominant cockroach species in homes in Tokyo areas, for asthma sensitization, we measured specific IgE antibodies to two cockroaches, P. fuliginosa and Blattella germanica, and to a mite, Dermatophagoides farinae, in 171 sera from children with asthma by Pharmacia's CAP system. We found that 16% of the sera had anti-P. fuliginosa IgE, whereas 9.9% had anti-B. germanica and 85% anti-D. farinae IgE. Further, we measured the levels of Per f I (Per a I equivalent) allergen in the house dust from living room, kitchen and bedding. We detected the allergen in eight of ten homes. The Per a I equivalent levels in kitchen were higher than in other sites, but they were much lower than Der I and Der II as Dermatophagoides allergens.
↩[68] - Eriksson NE, Moller C. Cockroach and Dermatophagoides pteronyssinus cross-react . J Allergy Clin Immunol 1996;98:471-472 ↩
[69] - Lodrup Carlsen KC, Carlsen KH, Buchmann MS, Wikstrom J, Mehl R Cockroach sensitivity in Norway: a previously unidentified problem? Allergy 2002;57:529-533
BACKGROUND: Little is known about cockroach sensitization in Scandinavia, whereas cockroaches are implicated in allergic diseases throughout large parts of the world. In association with the Genetics of Asthma International Network (GAIN) study, we report sensitization to cockroaches and possible association with IgE-mediated diseases in Norway.
METHODS: 100 Norwegian families (426 subjects) of 7-35-year-old sibling-pairs with asthma and their parents underwent questionnaire/interview (medical and exposure history), skin prick test (SPT) to common local inhalant allergens and German cockroach, and IgE specific to mites, mosquito, shrimp and cockroach. Cockroach sensitization was defined as positive if there was a positive (> or = 3 mm) skin prick test and/or presence of IgE antibody of class 2 or more.
RESULTS: Thirty-one subjects (7.5%) were sensitized (five monosensitized) to cockroach (27 by skin prick test and seven by IgE antibody, all with additional inhalant allergy). Co-sensitization was most common to grass (in 61%), cat (48%), dog (48%) and mites (42%). Reported allergic diseases in cockroach-sensitized subjects were asthma and rhinitis (n = 10), asthma only (n = 9), rhinitis only (n = 2) and neither asthma nor rhinitis (n = 10).
CONCLUSIONS: Since cockroach sensitization was relatively frequent in Norwegian atopic families, albeit with unclear clinical implications, we suggest that cockroach allergy should be considered in atopic subjects with respiratory disease.
↩[70] - Macan J, Plavec D, Kanceljak B, Milkovic-Kraus S. Exposure levels and skin reactivity to German cockroach (Blattella germanica) in Croatia. Croat Med J 2003;44:756-760
AIM: To evaluate German cockroach (Blattella germanica) allergen levels in house dust and skin reactivity to German cockroach in adult atopic and non-atopic subjects in inland areas of Croatia.
METHODS: Blattella germanica group 2 (Bla g 2) allergen was measured using the enzyme-linked immunosorbent assay (ELISA) on test strips (Dustscreen(TM) test, Heska AG, Switzerland) in 94 house dust samples collected from living room and bedroom floors, 35 from rural and 59 from urban areas. Skin prick testing with common inhalatory allergens, Blatella germanica, storage mites Lepidoglyphus destructor and Tyrophagus putrescentiae (ALK-Abello, Denmark) was performed in 187 adult outpatients, 131 from urban and 56 from rural areas. Total serum IgE levels were measured using the ELISA method. Subjects with relevant respiratory and/or skin symptoms, at least one positive skin prick test, and/or increased total IgE were considered atopic.
RESULTS: Positive skin prick test to cockroach was observed in 18/187 subjects (9.6%). The frequency of skin prick test positive subjects to cockroach was higher in atopic than in non-atopic subjects, but not significantly (12.2% vs. 4.7%; p=0.098). Of 15 atopic subjects with positive skin prick test to cockroach, 14 also had positive skin prick test to storage mites, 8 to house dust mites, 1 to pollens, and none to cockroach only. Positive skin prick test to storage mites was the only factor which significantly increased the risk for having positive skin prick test to cockroach (n=187; odds ratio, 109.82; 95% confidence interval, 2.06- 5853.5; p= 0.020). In all 94 house dust samples, Bla g 2 was not detectable.
CONCLUSIONS: Our results suggest that there is no relevant exposure to cockroach allergen in house dust samples from inland areas of Croatia. Consequently, positive skin prick test to cockroach is rare in adult subjects, even in atopics. Positive skin prick test to cockroach in atopics is in the majority of cases related to positive skin prick test to storage mites, probably due to cross-reactivity.
↩[71] - Adalsteinsdottir B, Sigurdardottir ST, Gislason T, Kristensen B, Gislason D. What Characterizes House Dust Mite Sensitive Individuals in a House Dust Mite Free Community in Reykjavik, Iceland? Allergol Int 2007;56:51-56
Background: Previous studies show that 6-9% of young adults in Reykjavik are sensitised to the house dust mite (HDM) Dermatophagoides pteronyssinus (D. pteronyssinus). However, only negligible amounts of HDM and HDM allergens were detected in their homes. The study investigates what characterizes these individuals.
METHODS: We investigated all participants in the European Community Respiratory Health Surveys I and II (ECRHS I and II) with D. pteronyssinus specific IgE, in the years 1991-92. A grass positive but D. pteronyssinus negative control group was recruited from the same cohort. A detailed questionnaire was administered and the specific IgE (Pharmacia CAP system) against six D. pteronyssinus cross-reactive allergens was measured.
RESULTS: Of 601 ECRHS I participants with available IgE results, 88% returned for ECRHS II, 8.4 years later. Of 49 individuals with D. pteronyssinus specific IgE in ECRHS I, 24 had become negative in ECRHS II. Compared with controls, HDM sensitive subjects were more often men who had lived on farms or kept aquaria fish in childhood. Of those with specific IgE against D. pteronyssinus in ECRHS I and II, 75% had detectable IgE antibodies (0.35kU/l) to cross-reactive allergens compared with none in the control group (p < 0.0001): Lepidoglyphus destructor (L. destructor) (67%), shrimp (58%), cockroach (33%), mosquito (17%), tropomyosin (17%) and blood worm (4%).
CONCLUSIONS: Icelanders with specific IgE to D. pteronyssinus are more often men who spent time on farms in childhood and today have high prevalence of IgE antibodies cross-reactive to D. pteronyssinus
↩[72] - Cunha L, Botelho C, Costa E, Lopes C, Falcao H. Evaluation of cockroach and shrimp sensitivity in a house dust mite allergic pediatric population. Allergy 2008;63(suppl. 88):168
Background: House dust mites (HDM) can present cross-reactivity with shellfish (shrimp) and other members of the Arthropoda family (ex: cockroach), being tropomyosin the panallergen more frequently involved.
Aims: Evaluate cutaneous sensitisation to cockroach and/or shrimp in a house dust mite allergic pediatric population followed in a specialized allergy clinic. Material and methods: The authors studied sequentially 164 children followed in a specialized allergy clinic, all allergic to HDM without history of food allergy. We performed SPT to aeroallergens [Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df), mixture of pollens from trees, grass and weeds; dog/cat dander and Blatella germanica (Alk Abellos)] and shrimp (Mercks). SPT were considered positive if the wheal size had a mean diameter _3 mm. Quisquare test and t-student test were used to perform statistical analysis.
Results: One-hundred and nine were male patients (66.5%) with mean age of 9.8 y (SD - 3.6). One-hundred and eight patients (65.9%) had familiar history of atopy. Respiratory symptoms were present in 149 patients (89%), in 16 (9.8%) they were associated to other allergic symptoms and 2 (1.2%) patients had only atopic dermatitis. One-hundred and one patients (61.3%) were under specific immunotherapy to HDM (mean follow-up of 20 months, SD of 23). sensitisation to cockroach and shrimp was present in 30 (18.3%)/31 (18.9%) of patients, respectively. All patients sensitised to cockroach or shrimp had respiratory symptoms (asthma and/or rhinitis). The mean wheal diameter of the SPT to Dp/Df in patients sensitised or not to cockroach had no statistical differences (P=0.06/P=0.14). The same results were found when we compared the mean wheal diameter of the SPT to Dp/Df and presence or not of sensitisation to shrimp (P=0.055/P=0.23).
Conclusions: Simultaneous sensitisation to HDM and cockroach or shrimp is present in 18% of patients. Co-sensitisation to those three allergens appears in only 7% of patients. In this pediatric population, cross-reactivity was not considered determinative.
↩[73] - Witteman AM, van den Oudenrijn S, van Leeuwen J, Akkerdaas J, van der Zee JS, Aalberse RC. IgE antibodies reactive with silverfish, cockroach and chironomid are frequently found in mite-positive allergic patients. Int Arch Allergy Immunol 1995;108:165-169
Approximately 30% of the house dust mite allergic patients in The Netherlands have IgE antibodies reactive with silverfish, cockroach and/or chironomid. In allergic patients without IgE antibodies against Dermatophagoides pteronyssinus less than 5% have IgE antibodies reactive with these insects. By means of RAST inhibition studies it is shown that cross-reactivity exists between D. pteronyssinus and silverfish, cockroach or chironomid. This means that a positive RAST for silverfish, cockroach, chironomid or D. pteronyssinus cannot be taken as evidence for exposure.
↩[74] - Riario-Sforza GG, Della Torre F, Antonicelli L, Bonifazi F, Giordano T, D'Amato G, et al. Sensitization to cockroach in Italy: a multicentric study. Allergy Asthma Proc 1997;18:23-28
The prevalence of sensitization to cockroach (CR) was evaluated by skin prick test (SPT) in 1299 patients consecutively referred for rhinitis and asthma to five allergy centers in northern, central, and southern Italy. In patients with positive SPT to CR, an SPT with D. pteronyssinus extract was made. RAST inhibition was performed with pooled sera of subjects with positive SPT and RAST of at least Class 3 for CR and D. Pteronyssinus, to assess the cross-reactivity between these two allergenic sources. The protein content of allergen extracts was assayed by isoelectric focusing (IEF). A total of 170 patients (13%) had a positive SPT with CR extract, and 133 of them (78.2%) were also positive for D. pteronyssinus. RAST inhibition showed a cross-reactivity between CR and D. pteronyssinus, and IEF detected in the extracts employed a series of bands focusing at the same pI of CR and D. pteronyssinus relevant allergens. Therefore, the prevalence of sensitization to CR in the population studied, corresponding to 13%, suggests that CR extract might be added to standard SPT panels for respiratory allergy. However, the cross-reactivity with D. pteronyssinus calls for careful evaluation of the clinical importance of this sensitization.
↩[75] - Jeong KY, Hwang H, Lee J, Lee IY, Kim DS, Hong CS, et al. Allergenic characterization of tropomyosin from the dusky brown cockroach, Periplaneta fuliginosa. Clin Diagn Lab Immunol 2004;11:680-685
Household arthropods are one of the most common causes of allergic diseases. Four species of cockroaches are found to reside in Korean homes, but published work deals almost exclusively with the German and American cockroaches. This study was undertaken to investigate the cross-reactive allergenic components of the dusky brown cockroach, Periplaneta fuliginosa. Enzyme-linked immunosorbent assay (ELISA) inhibition and immunoblot analyses for the dusky brown cockroach were performed with Blattella germanica and Dermatophagoides farinae allergic sera. cDNA encoding tropomyosin, which is a well known cross-reactive pan-allergen, was cloned by reverse transcriptase PCR, and recombinant protein was produced by using a pET-28b expression system. Native tropomyosin was purified by ammonium sulfate fractionation and electroelution. The immunoglobulin E (IgE) reactivities of native and recombinant tropomyosins were compared by an ELISA inhibition study. All 30 sera tested showed P. fuliginosa-specific IgE, and the IgE-binding reactivity of the P. fuliginosa extract was inhibited as much as 79.4% by a B. germanica extract and as much as 63.3% by a D. farinae extract. The deduced amino acid sequence of cloned cDNA was identical with that of Periplaneta americana tropomyosin (98.5% nucleotide sequence identity). Seven of 26 (26.9%) allergic sera had IgE specific for recombinant protein, and the maximum inhibition of P. fuliginosa-specific IgE achieved with recombinant tropomyosin was 37.7% at an inhibitor concentration of 10 microg/ml. Native tropomyosin inhibited the binding of IgE to the P. fuliginosa, B. germanica, and D. farinae extracts by 65.0, 51.8, and 39% at an inhibitor concentration of 1 microg/ml. P. fuliginosa appears to possess allergens that are highly cross-reactive with allergens of B. germanica and D. farinae. Tropomyosin was found to be a major allergenic component accounting for the cross-reactivity between cockroaches and dust mites.
↩[76] - Eriksson NE, Moller C. Cockroach and Dermatophagoides pteronyssinus cross-react . J Allergy Clin Immunol 1996;98:471-472 ↩
[77] - Satinover SM, Reefer AJ, Pomes A, Chapman MD, Platts-Mills TAE, Woodfolk JA. Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 2005;115:803-809
BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied .
OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects .
METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques .
RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon .
CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.
↩[78] - Sidenius KE, Hallas TE, Poulsen LK, Mosbech H. Allergen cross-reactivity between house-dust mites and other invertebrates. Allergy 2001;56:723-733 ↩
[79] - Arruda LK. Cockroach allergens. Curr Allergy Asthma Rep 2005;5:411-416
Cockroaches produce several potent allergens associated with sensitization and the development of asthma. Many of these allergens have been cloned and produced as biologically active recombinant proteins. Three-dimensional structure and molecular modeling, in addition to studies on the biological functions of these proteins in the cockroach, have provided new insights into the ability of these allergens to cause immunoglobulin E antibody responses. Recent studies have pointed to a potential role of recombinant cockroach allergens for diagnosis and the development of novel therapeutic strategies that may benefit cockroach-allergic patients.
↩[80] - Satinover SM, Reefer AJ, Pomes A, Chapman MD, Platts-Mills TAE, Woodfolk JA. Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 2005;115:803-809
BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied .
OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects .
METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques .
RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon .
CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.
↩[81] - Purohit A, Shao J, Degreef JM, van Leeuwen A, van Ree R, Pauli G, et al. Role of tropomyosin as a cross-reacting allergen in sensitization to cockroach in patients from Martinique (French Caribbean island) with a respiratory allergy to mite and a food allergy to crab and shrimp. Eur Ann Allergy Clin Immunol 2007;39:85-88
BACKGROUND: Tropomyosin has been described as cross-reacting allergen between mite, cockroach and shrimp.
METHODS: In 13 patients with asthma and/or rhinitis sensitized to mite and/or German cockroach and presenting urticaria, oral allergy syndrome or angio-edema upon eating shrimp and/or crab, we measured specific IgE to mite, cockroach, crab and shrimp tropomyosin.
RESULTS: Ten patients had specific IgE to tropomyosin from mite, 8 from shrimp, 6 from crab and 5 from cockroach. AST inhibition tests indicated that mite allergen is a primary sensitizer and is cross-reacting with shrimp, crab and cockroach allergens. CONCLUSION: Tropomyosin could be the cross-reacting allergen relevant for clinical symptoms to mite, cockroach, shrimp and crab.
↩[82] - Reder MW, Wild LG, El-Dahr JM, Villacis J, Demerell D, Soteres D, et al. IgE Antibody Activity of Shrimp Allergic Subjects to Inhalant and Food Allergens. J Allergy Clin Immunol 2005;115(2 suppl.):S92
RATIONALE: Shrimp is an important allergenic seafood. The major shrimp allergen is the muscle protein tropomyosin. Since tropomyosin is present in other inhaled and ingested allergens, it was of interest to determine potential sensitivity of shrimp allergic subjects to these other allergens
METHODS: Shrimp allergic subjects were recruited by advertisement 58 subjects (25 male, 33 female) ages 18-63, with definitive histories of shrimp allergy, as determined by questionnaire, were selected. Subjects were skin prick tested (ST) with aeroallergen and shellfish food extracts from Hollister-Stier; positive ST was determined by wheal with flare. IgE antibody responses to shrimp were measured by CAP-RAST
RESULTS: 37/58 subjects were ST positive to at least 1 shrimp extract (64%). 41 (71%) were positive to either crab, lobster, crawfish, or oyster, 51 (88%) to either cockroach or DM, 19 (33%) to tree pollens, and 14 (24%) to grass pollens. Of the 37 subjects ST positive for shrimp, 35 (96%) were positive to crab, lobster, or crawfish. 36 (97%) reacted to either cockroach or DM. Of the 58 original subjects, 28 (48%) had undetectable IgE to shrimp (class 0) on CAP-RAST analysis. Of the 37 shrimp ST positive subjects, only 10 (27%) had undetectable IgE to shrimp
CONCLUSIONS: Shrimp allergic subjects show significant reactivity to other shellfish, the greatest was to lobster, the least to oysters. Significant reactivity was observed to cockroach and DM extracts which is probably due in part to cross reactivity of invertebrate tropomyosin allergens. Better understanding of this should help improve diagnosis and therapy of food allergy
↩[83] - Santos AR, Rodrigues MC, Thiesen M, Zampolo AS, Ferriani VPL, Arruda L. Use of Recombinant Proteins for Diagnosis of Cockroach Allergy in Patients with Asthma and/or Rhinitis Living in Brazil. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°843
RATIONALE: Several cockroach allergens have been produced as recombinant proteins, however skin test reactivity to these allergens has been established in a limited number of patients
METHODS: Frequency of positive skin tests to recombinant allergens of Blattella germanica (rBlag 2, rBlag 4, rBlag 5) and Periplaneta americana (rPera 1 and rPera 7) was evaluated in 50 cockroach allergic patients with asthma and/or rhinitis, aged 7 to 70 years-old. Patients presented positive skin tests to P. americana and B. germanica. Skin prick tests with recombinant allergens were carried out at 10mcg/ml or 5mcg/ml (rPera 1) concentrations. Recombinant tropomyosins from P. americana (rPera 7) and A. lumbricoides, expressed in Pichia pastoris, were used in chimeric ELISA to quantitate IgE antibodies in sera of 48/50 patients
RESULTS: Positive skin tests to rBlag 2, rBlag 4, rBlag 5, rPera 1 and rPera 7 were found in 4(8%), 3(6%), 3(6%), 3(6%), and 18(36%) patients, respectively. Positive test to at least one allergen was found in 46% of patients. IgE antibodies to P. americana and A. lumbricoides tropomyosins were detected in 26(54.2%) and 25(52%) patients, respectively Levels of IgE to rPer a 7 varied from 0.6 to 200IU/mL (geometric mean GM 2.3IU/mL) and to A. lumbricoides tropomyosin, from 0.6 to 500IU/mL (GM 1.8IU/mL). There was a significant correlation of levels of IgE to Ascaris and P. americana tropomyosin (r=0.41; p=0.0034)
CONCLUSIONS: Recombinant allergens could be safely used for diagnosis of cockroach allergic patients living in Brazil. This approach could be improved by inclusion of additional cockroach allergens in the panel Funding:
↩[84] - Arruda LK, Santos ABR, Sales VSF, Ferriani VL, Dantas VCR, Rodrigues CEB, et al. Tropomyosin in Ascaris Lumbricoides: A Cross-Reactive IgE Binding Protein ? AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°378
RATIONALE: It is estimated that 1.4 billion people are presently infected with Ascaris lumbricoides. We have shown that infection with A. lumbricoides is an important risk factor for wheezing in children living in Brazil, and that tropomyosin could be involved in cross-reactivity in invertebrates. We aimed to determine the sequence of A. lumbricoides tropomyosin, and to investigate IgE antibody responses to tropomyosins from Ascaris and cockroach in children from a parasite-endemic area.
METHODS: Primers based on the sequences of cockroach and Anisakis simplex tropomyosin were used in RT-PCR. IgE antibodies to tropomyosins from A. lumbricoides and cockroach were quantitated in sera of 146 preschool children by chimeric ELISA.
RESULTS: The sequence of the 284 amino acid protein from A. lumbricoides showed 90% to 98% identity to tropomyosins from other parasites, including Anisakis simplex, and 74% and 69% identity to mite and cockroach tropomyosins, respectively. IgE to tropomyosin was found in 77/146 (52.7%) children, and there was a significant correlation of levels of IgE to A. lumbricoides and P. americana tropomyosin (r=0.8, p<0.0001). Total IgE levels were significantly higher in the group with IgE to tropomyosin as compared to the group with no IgE to tropomyosin (GM: 904 and 409 IU/mL, p=0.006). No significant differences were observed in the frequency of positive stools for Ascaris in both groups (egg counts 0 to 61,608 eggs/g of feces).
CONCLUSIONS: A. lumbricoides tropomyosin is a major IgE binding protein in children from parasite-endemic areas.
↩[85] - Santos ABR, Rocha GM, Oliver C, Ferriani VPL, Lima RC, Palma MS, et al. Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach. J Allergy Clin Immunol 2008;121:1040-1046
BACKGROUND: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma .
OBJECTIVE: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides .
METHODS: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program .
RESULTS: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6 .
CONCLUSION: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.
↩[86] - Arruda LK. Tropomyosin in Parasites - A Crossreactive IgE-binding Protein ? Allergy Clin Immunol Int 2005;17:243-245
Background: The role of infections with intestinal parasites in the development of allergy and asthma is still controversial, with some studies pointing towards protective effects of parasitic infection and others suggesting a heightened risk of allergic sensitization. Methods/Data base: A review of the literature.
Results: Tropomyosins from mites, cockroach, shrimp, and parasites including Ascaris lumbricoides show a high degree of sequence identity. Studies on IgE-binding epitopes have provided molecular evidence for crossreactivity among these invertebrate tropomyosins, which might have clinical relevance.
Conclusion: Several issues including age at the time of initial parasitic infection, parasite load, type of parasite, socioeconomic conditions, life-style, and environmental allergen exposure may play a role in the complex relationship between parasite infections and the development of allergic diseases. Our results prompted us to speculate that crossreactive allergens such as tropomyosin at the time of the initial Ascaris infections could facilitate subsequent development of crossreactive IgE antibody responses upon exposure to mite or cockroach, which could lead to airway inflammation and asthma. Therefore, infection with Ascaris could have an adjuvant effect on the development of asthma, in the subset of Ascaris-infected individuals who develop IgE responses to tropomyosin.
↩[87] - Santos ABR, Rocha GM, Oliver C, Ferriani VPL, Lima RC, Palma MS, et al. Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach. J Allergy Clin Immunol 2008;121:1040-1046
BACKGROUND: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma .
OBJECTIVE: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides .
METHODS: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program .
RESULTS: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6 .
CONCLUSION: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.
↩[88] - Patterson ML, Slater JE. Characterization and comparison of commercially available German and American cockroach allergen extracts. Clin Exp Allergy 2002;32:721-727
BackgroundIn this study we examine the variability among unstandardized cockroach allergen extracts. MethodsWe obtained 24 aqueous and glycerinated cockroach allergen extracts from nine manufacturers. We used previously characterized cockroach extracts, E2-Cg and E2-Ca, as references. The modified ninhydrin assay was used to determine protein concentration of each extract. Relative potencies of extracts were determined by competition ELISA, using a human allergic serum pool. Bla g 1 and Bla g 2 levels of glycerinated German cockroach extracts were determined by ELISA using monoclonal antibodies. Extracts were also analysed by SDS-PAGE. ResultsCommercial cockroach allergen extracts had highly variable protein contents that were lower than the protein contents of the references. Electrophoretic data confirmed the presence of a variable number and intensity of protein bands in extracts among manufacturers. The relative potencies of the commercial extracts were between 10 and 782BAU/mL for German cockroach and 10-250BAU/mL for American cockroach. The mean Bla g 1 content of the commercial extracts was significantly lower than that of the reference (P=0.001). The mean Bla g 2 content of the commercial extracts was higher than that of the E2-Cg reference but the Bla g 2 levels were more variable compared to Bla g 1. In glycerinated German cockroach extracts, protein concentrations, relative potencies and specific allergen levels were significantly correlated (P<0.001). ConclusionOur tests indicate that commercially available cockroach allergen extracts are variable in protein content, electrophoretic banding patterns, relative potency and Bla g 2 levels. In glycerinated German cockroach extracts, protein concentrations, relative potencies and specific allergen levels were significantly correlated.
↩[89] - Sudha VT, Srivastava D, Arora N, Gaur SN, Singh BP. Stability of Protease-Rich Periplaneta americana Allergen Extract During Storage: Formulating Preservatives to Enhance Shelf Life. J Clin Immunol 2007;27:294-301
Allergenic proteins in extracts degrade rapidly and lose potency on storage. Hence, formulation of optimum conditions is required to enhance shelf life of extracts for proper allergy diagnosis and immunotherapy. In the present study, allergenic potency of P. americana proteins was evaluated after storage with epsilon-aminocaproic acid (EACA), sucrose, glycerol, pepstatin A, and aprotinin, individually for 1, 3, 6, and 12 months at 4, 25, and 37 degrees C. P. americana extract stored with EACA and sucrose individually retained potency comparable to proteins in standard extract (freeze-dried extract, stored at-70 degrees C) upto 6 months at 4 degrees C. The extracts without preservatives or with glycerol, pepstatin A, aprotinin, or stored at 37/25 degrees C were severely degraded and lost potency by 3 months. A formulation containing a combination of EACA and sucrose enhanced the shelf life of P. americana proteins upto 12 months at 4 degrees C. Hence, EACA and sucrose together show better potential for stabilization of protease-rich extracts.
↩[90] - Sidenius KE, Hallas TE, Poulsen LK, Mosbech H. Allergen cross-reactivity between house-dust mites and other invertebrates. Allergy 2001;56:723-733 ↩
[91] - Thangam Sudha V, Arora N, Sridhara S, Gaur SN, Singh BP. Biopotency and identification of allergenic proteins in Periplaneta americana extract for clinical applications. Biologicals 2007;37:131-137
Commercial cockroach extracts for diagnosis and therapy show batch-to-batch variation. This study aimed to standardize Periplaneta americana extract based on major IgE binding components using hypersensitive patients' sera. Extracts were prepared in phosphate buffered saline (PBS) or NH(4)HCO(3), from freeze-dried or 37 degrees C dried material and compared with commercial extracts by immunobiochemical methods. Cockroach positive patients' sera were collected after intradermal tests and specific IgE enzyme linked immunosorbent assay (ELISA). Allergenic proteins were identified by western-blotting and potency of extracts determined by ELISA-inhibition. Adult P. americana extract from freeze dried source material in PBS (PA extract) resolved into 45 protein bands and showed 22 IgE binding components with pooled patients' sera. It required 9-12ng self-proteins for 50% ELISA-inhibition. Individual patients' sera identified 23, 28, 35, 38, 40, 49, 72, 78 and 97kDa as major IgE binding components in PA extract. Nymph extract exhibited similar potency and protein profile to PA extract with 72 and 78kDa proteins present in high intensities. Commercial extracts exhibited only 6-11 IgE reactive bands compared to PA extract and required 40 folds or more protein for 50% ELISA-inhibition. PA extract from freeze-dried source material seems a potent allergen preparation with 9-major IgE binding components. It can be referred to upgrade the quality of commercial extracts exhibiting low potencies due to poor quality source material, inadequate extraction procedures and improper storage.
↩[92] - Arruda LK, Jorge PPO, Toledo EC, Santos ABR, Rodrigues MC, Ferriani VPL, et al. Recombinant Allergens for Diagnosis of Mite and Cockroach Allergy in Brazilian Children. J Allergy Clin Immunol 2008;121:S177
RATIONALE: Recombinant allergens have been shown to induce positive skin tests in patients with asthma and/or rhinitis. The use of recombinant allergens for diagnostics has not been investigated in a large, less selected population.
METHODS: A group of 531 school children 13-14 years-old was selected among 4498 children evaluated by the ISAAC questionnaire, using a 1:2 ratio of presence and absence of wheezing in the past year. Children underwent skin testing with commercial inhalant extracts, and recombinant allergens derived from mites (rDerp1, rDerf1, rDerp2, rDerp5, and a mixture containing all four allergens) and cockroach (rPera1, rPera7, rBlag2, rBlag4, rBlag5) at 10 mcg/mL concentration. Skin tests were considered positive when a wheal of equal or greater than 4 mm mean diameter, accompanied by erythema, developed 15 minutes following allergen application.
RESULTS: Presence of a positive skin test to mites, cockroach, cat and dog; to the mite recombinant mix, and to each of the recombinant allergens except rPera1 (reactivity 1.3%) was significantly associated with current wheezing. Kappa analysis revealed a strong concordance of results of skin tests using commercial mite extract and recombinant mite mix (index of 0.7591);and a moderate concordance using Periplaneta americana extract and recombinant P. americana allergens (index of 0.4637). Among 251 children with positive skin tests to D. pteronyssinus, 190(75.7%) had positive tests to the recombinant mix, and only 2/280 with negative tests to D.
pteronyssinus presented positive reactions to the recombinant allergens.
CONCLUSIONS: Mixtures of recombinant mite allergens can be safely and effectively used for the diagnosis of mite allergic patients.
↩[93] - Pomés A, Li M, Wünschmann S, King EM, Alexandratos J, Chapman MD, et al. The Crystal Structure of the Complex of Bla g 2 with an Antibody Fragment Reveals Cation-pi and Sugar Interactions. J Allergy Clin Immunol 2008;121:S213
RATIONALE: The interactions between the cockroach allergen Bla g 2, homologous to aspartic proteases, and a monoclonal antibody (mAb) whose epitope is involved in IgE antibody binding were analyzed by crystallography.
METHODS: Purified recombinant Bla g 2 was co-crystallized with Fab‚ fragments of the mAb 7C11. The structure was solved by molecular replacement and refined using the amino acid sequence obtained by Reverse-Transcription-PCR of mRNA from the mAb-producing cell line.
Antibody binding to Pichia pastoris expressed mutants was performed by multiplex technology.
RESULTS: Bla g 2 interactions with the mAb among others include a continuous epitope (residues 60-70 plus Arg83), recognized predominantly by the light chain, and are driven by a number of charged residues. Lys65, Arg83 and Lys132 are involved in strong cation-pi interactions with Tyr53, Tyr92 (light chain) and Tyr33 (heavy chain), respectively.Mutations of these cationic residues affected mAb and/or IgE antibody binding.
Carbohydrate moieties of the glycosylated allergen molecules are involved in crystal contacts with the mAbs through the trimannosyl core, common between N-linked oligosaccharides of the natural and recombinant allergens.
Lys24 and Asp70, and Asp110 and Lys199 from the light chain, interact with the N-acetyl-glucosamines from the sugars in Asn268 and Asn317, respectively. The third glycosylation site (wild type N93) is expected to also interact with the mAb, since Ser85 from heavy chain binds to the mutated Gln93.
CONCLUSIONS: Analysis of the allergen-antibody interactions from the crystal structure of a Bla g 2-Fab‚ complex enables a rational approach for the design of allergen molecules with reduced IgE antibody binding.
↩[94] - Holmquist I, Jortso L, Kober A, Sjolander S. Investigation of cross-reactions and co-sensitisations patterns in cockroach sensitized individuals. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°318
Background: IgE sensitisation to cockroach is often associated with multi-sensitisation to a number of allergens, which may complicate the evaluation of IgE antibody (ab) responses to cockroach. Concurring responses to cockroach, house dust mite, shrimp and bromelain (BR) are often seen. Mite, shrimp and cockroach are known to share the allergen tropomyosin (TM) and cross-reactions have been demonstrated. BR has been used as an indicator of IgE ab to cross-reacting carbohydrate determinants (CCD) and indications of their presence on cockroach allergens has been demonstrated.
METHODS: 730 human sera with known IgE ab responses to cockroach were analysed for responses to mite and shrimp, using the ImmunoCAP System. 84 of the sera were analysed for responses to BR and ImmunoCAP RAST-inhibition in 20 sera by the use of a BR based CCD inhibitor was performed.
RESULTS: Simultaneous responses to cockroach and mite/shrimp were found in 83% and 67% of the sera, respectively. IgE ab responses to mite were significantly higher than responses to cockroach (p<0.001) although no significant correlation was seen. No significant difference was demonstrated between response levels to shrimp and cockroach, but a strong correlation (p<0.05) was shown. The response patterns indicate that simultaneous responses to cockroach and mite, beside the known cross-reaction to TM, may comprise additional co- or cross-mechanisms. Co-exposure of mite and cockroach has been demonstrated earlier and co-sensitisation therefore appears likely. The response pattern also indicates that simultaneous responses to cockroach and shrimp to a high degree are represented by cross-reactions to, for example, TM. Finally, a significant response to BR was found in 74% of the sera. Inhibition using a CCD inhibitor showed inhibition in 80% of the sera. The results strongly indicate the presence of CCD on cockroach allergens and that cross-reactions of this type seem to occur frequently.
CONCLUSION: In this study we describe concurrent IgE responses to cockroach, mite and shrimp, and suggest that they either are due to co-sensitisation (mite) and/or cross-reactions specific for TM (mite and shrimp). We also propose the presence of recurrent cross-reactions to CCD. Awareness of these patterns will facilitate the evaluation of IgE responses to cockroach, and concomitant testing for several allergens and/or components may therefore be a useful tool in the subsequent diagnosis of cockroach allergy.
↩[95] - Pevec B, Pevec MR, Markovic AS, Batista I. IgE cross-reactivity between cockroach, birch pollen, and insect venom allergens. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°35 ↩
[96] - 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
Imprimer la bibliographieBACKGROUND: 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.
↩
