Abeilles, guêpes, frelons

E pour Europe, Am pour Amérique, As pour Asie
Titre
	Français	Anglais US	Anglais UK	Espèces
Apides _	Abeilles	Honeybees	Honeybees	Apis mellifera Apis cerana (As) A. mellifera scutellata (Am)
Bourdons	Bumblebees	Bumblebees	Bombus terrestris (E) Bombus pennsylvaticus (Am)
Vespides	Guêpes	Paper wasps	Wasps	Polistes (AM) = exclamans, apachus, fuscatus, annularis,… Polistes (E) = dominulus, gallicus, nimpha,…
Guêpes	Yellow jackets	Wasps	Vespula (Am) = maculifrons, flavopilosa, squamosa, vidua, vulgaris, etc... Vespula (E) = vulgaris, germanica, rufa, …
Guêpes		Wasps	Dolichovespula (E) = media, sylvestris, saxonica, …
Frelons	Hornets		Dolichovespula (Am) = arenaria, maculata
Frelons	Hornets	Hornets	Vespa (E) = crabro, orientalis Vespa mandarinia (Am), …

	Double positivité
	En CAP	En IDR
Présence d’IgE anti-CCD	93 % des sujets	76 %
Absence d‘IgE anti-CCD	13 %	32 %

	Abeille	Guêpe
Insecte selon les patients	100	100
Positifs en IDR à 10-4	26 pour la guêpe	39 pour l’abeille
Avec un CAP > 0,7 kU/l	50 pour la guêpe	41 pour l’abeille
CAP broméline positif	52	13
rApi m 1 positif	96	17
rVes v 5 positif	17	96

[1] - Birnbaum J. Allergie aux venins d'hyménoptères. Qui, comment et combien de temps désensibiliser ? Rev Fr Allergol Immunol Clin 2005;45:489-492

Si la sensibilisation aux venins d'hyménoptères est fréquente, l'allergie est plus rare mais se retrouve chez 1 à 3 % de la population générale. Sa gravité clinique, tel le choc anaphylactique, est l'indication immédiate d'une désensibilisation. Les manifestations légères cutanéomuqueuses ne relèvent pas a priori d'une désensibilisation. La connaissance de facteurs de risque à la récidive ou à l'aggravation d'une nouvelle réaction en cas de piqûre permet d'adapter l'indication à chaque patient. Quel que soit le protocole de désensibilisation utilisé, en sachant que les protocoles rush ou ultrarush sont actuellement les plus prescrits et conseillés, des réactions secondaires sont observées. Peu de facteurs sont connus comme permettant d'identifier le patient à risque d'une mauvaise tolérance lors de la désensibilisation. Les antihistaminiques ne préviennent que les réactions locorégionales et les réactions générales minimes. Leur rôle éventuel comme agent pouvant augmenter l'efficacité de la désensibilisation les fait conseiller pendant les phases de progression des doses de venin. En dehors de certaines situations, la dose de rappel doit être au minimum de 100 µg mais parfois elle devra être de 200 µg. La durée de la désensibilisation sera au minimum de cinq ans. Elle pourra être arrêtée au bout de trois ans si le bilan biologique (tests cutanés et IgEs) est négatif et sera poursuivi au-delà de cinq ans dans certaines situations à risque.

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[2] - Bilò BM, Bonifazi F. Epidemiology of insect-venom anaphylaxis. Curr Opin Allergy Clin Immunol 2008;8:330-337

PURPOSE OF REVIEW: Knowledge of the epidemiology, natural history and risk factors of insect-venom allergy is crucial for improving the clinical management of allergic patients. This review focuses on the recent research on these aspects of Hymenoptera-sting anaphylactic reactions. RECENT FINDINGS: The latest data from population-based studies of anaphylactic reactions due to Hymenoptera stings, as well as those extrapolated from studies of epidemiology of anaphylaxis due to any cause are reviewed. The topic of biphasic anaphylactic reactions due to insect stings is also examined. Although no parameter has been identified that can predict which sensitized individuals will have a future anaphylactic reaction, several factors associated with the severity of a systemic resting reaction are known and emphasized here. SUMMARY: As half of individuals with fatal sting reactions had no documented history of previous systemic reaction, we need to further improve the knowledge of the natural history and risk factors, especially in asymptomatic-sensitized individuals. Moreover, and no less important, the epidemiological studies on sting anaphylaxis conducted in the 2000s continue to reveal the poor management of allergic patients and the startling lack of awareness of the efficacy of venom immunotherapy. These findings indicate the urgent need to educate the general population and doctors on the management of venom-allergic patients.

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[3] - Müller U, Golden DB, Lockey RF, Shin B. Immunotherapy for hymenoptera venom hypersensitivity. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 377-92

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[4] - Panzani RC, Ariano R. Arthropods and invertebrates allergy (with the exclusion of mites): the concept of panallergy. Allergy 2001;56(suppl. 69):1-22

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[5] - Rudeschko O, Machnik A, Dörfelt H, Kaatz HH, Schlott B, Kinne RW. A novel inhalation allergen present in the working environment of beekeepers. Allergy 2004;59:332-337

BACKGROUND: Inhalation allergies, caused by allergens from various kinds of pollen, house dust mites, animal epithelium, and mould fungi, are strongly increasing in frequency. In 2.6% of the cases the allergen source remains unidentified. The present paper describes a so far unknown inhalation allergy which was observed in the case of a patient working with hives. METHODS AND RESULTS: The allergen was characterized by immunoblotting, enzyme-linked immunosorbent assay inhibition, and isoelectrofocusing, using the serum of the patient. It is present in both the bee bodies and the larvae, has a molecular mass of 13 kDa, and an isoelectric point of 5.85. It is thermolabile and does not cross-react with allergens from birch, mugwort and timothy grass pollen, mould fungi, or bee venom. The N-terminal amino acid sequence of allergen from larvae was determined to be (2)QIEELKTRLHT(12). A similar allergen of 13 kDa was also found in Varroa mite accompanying bee populations . CONCLUSION: Honey bees (including the larva stadium) and Varroa mite contain a 13-kDa protein causing an allergic reaction. Presently, there is no evidence whether the case described is a singular phenomenon or whether this allergen is a more common inducer of allergies among subjects exposed to honey bees. However, a bee and Varroa mite allergy has to be considered for beekeepers after exclusion of known inhalation allergies.

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[6] - Armentia A. Adverse reactions to wine: think outside the bottle. Curr Opin Allergy Clin Immunol 2008;8:266-269

PURPOSE OF REVIEW: Wine contains chemical and biological contaminants. Symptoms such as facial flushing, asthma and oral allergic swelling and burning (oral syndrome) have been attributed to these contaminants and food additives. Their clinical implications should be known. RECENT FINDINGS: Recent studies have reported a high prevalence of hypersensitivity symptoms after intake of alcoholic drinks in the general population. Red wine was the most common beverage implicated. Wine contains many contaminants. Some of them come from Hymenoptera insects that fall into the wine when grapes are collected and pressed. We have found patients with allergic symptoms related to wine consumption who are sensitized to Hymenoptera venom without previous stings. The aim of this study is to assess the potential importance of their sensitization to Hymenoptera antigens as the cause of their symptoms and also to comment on other recent studies on wine hypersensitivity. SUMMARY: We found patients with allergic symptoms related to wine consumption who are sensitized to Hymenoptera venoms. Challenges were negative with sulfites, other additives and aging wines, but positive with young wines. Sera from all the patients detected Hymenoptera venom antigens. We report the first cases of sensitization to venom antigens by the oral route.

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[7] - Cretin JY. Hyménoptères d'Europe et du bassin méditerranéen. Rev Fr Allergol Immunol Clin 2006;46:274-276

Parmi les hyménoptères, seuls les aculéates (latin aculeatus « muni d'un aiguillon ») sont susceptibles d'infliger des piqûres grâce à leur dard venimeux. Tout en restant dans le domaine européen, la diversité spécifique de ce groupe est passée rapidement en revue, avec un rappel des familles classiquement connues pour provoquer des réactions allergiques (insectes sociaux : apidés, vespidés, formicidés), et une évocation des familles non sociales qui, fortes de plusieurs milliers d'espèces bien plus discrètes, entrent parfois en contact accidentellement avec l'homme. Il est en effet hautement probable que certaines réactions atypiques ou non reliées ensuite à des venins connus soient dues à ces hyménoptères solitaires, rencontrées fortuitement au cours des diverses activités humaines, à la campagne comme dans les secteurs fortement urbanisés.

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[8] - Pravettoni V, Mauro M, Bertolotti F, Primavesi L, Piantanida M, Labardi D, et al. Hymenoptera venom allergy: identification of a new venom allergen responsible of Xylocopa violacea anaphylaxis. Allergy 2008;63(suppl. 88):80

Background: Carpenter Bee (Xylocopa violacea) is a solitary, fast flight, ubiquitous bee usually non aggressive and rarely stinging. Up to now, in medical literature Carpenter bee anaphilaxis has never been reported. In this study we described two cases of Xylocopa violacea venom anaphylaxis and we determined the relevant allergens of Xylocopa violacea venom. Methods: We report the case history of two patients (A, 26 y.o and B, 39 y.o.) with anaphylactic systemic reaction after Xylocopa violacea sting. All the two patients were also stung by yellow jacket before and after anaphylaxis, well tolerating the stings. At the clinical visit we performed serum venom specific IgE antibodies and venom skin prick test and intradermal tests with yellow jacket YJ, paper wasp W, European hornet EH, honeybee HB and Polistes dominulus venoms. Five patients with negative allergenic history were used as negative controls. Xylocopa violacea extract was supplied by Anallergo (Florence, Italy). SDS-PAGE and Immunoblotting: SDSPAGE was performed with Xylocopa violacea and all the above mentioned venoms. The gel was blotted on a nitrocellulose membrane, cutted into strips and matched with the sera of the two patients. After incubation with anti-IgE antibodies, the allergenic bands were detected by a chemiluminescence revealing system. Results: In the two allergic patients skin tests and serum IgE detection were positive only for yellow jacket extract. In both the patients in vitro analysis did not show any IgE binding in the honeybee immunoblotting, while IgE reactive bands at 25.2 kDa, 19.1 kDa and 6.5 kDa were found in the Xylocopa violacea extract. The 25.2 kDa allergen was also recognised in EH and YJ venoms, representing a possible cross reactive allergen between Xylocopa and Vespid venoms, surely not responsible of anaphylaxis, as the two patients were stung by yellow jacket without experience of systemic reactions. No response was found in control sera. The band at 19.1 kDa seemed to be specific of Xylocopa violacea venom. The band at 6.5 kDa, corresponding to mellitin, surprisingly presented an IgE reactivity only in Xylocopa violacea extract, suggesting a difference in HB and Xy mellitin, despite the belonging of these two Hymenoptera to the Apidae family. Conclusion: In these two cases of Xylocopa violacea venom anaphylaxis the responsible allergen seems to be the protein at 19.1 kDa, only cross inhibition study could confirm this finding

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[9] - Hoffman DR. Structural biology of allergens from stinging and biting insects. Curr Opin Allergy Clin Immunol 2008;8:338-342

PURPOSE OF REVIEW: Modern techniques in genomic and protein research are applied to the study of stinging and biting insect allergens. RECENT FINDINGS: Three-dimensional structures of additional insect venom and salivary allergens have been determined. An approach to determining B-cell epitopes has been used for hyaluronidase. A number of new venom and salivary allergens have been characterized. The structures and significance of several insect allergens have been updated. Investigations continue into distinguishing venom crossreactivity from multiple sensitization. Further studies are clarifying the significance of carbohydrate epitopes. Genomic and proteomic techniques are being used in the investigation of proteins and peptides in insect venom and saliva. SUMMARY: The nature of venom crossreactivity and the B-cell and T-cell epitope structures of insect venom and salivary allergens are beginning to be elucidated

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[10] - King TP, Guralnick M. Hymenoptera 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. 237-49

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[11] - Hoffman DR. Allergens in hymenoptera venom. XXVI: The complete amino acid sequences of two vespid venom phospholipases. Int Arch Allergy Immunol 1994;104:184-190

The 2 major allergenic proteins in vespid venoms are antigen 5s and phospholipases (PLs). Vespid PLs have a molecular weight of about 34,000 and have been previously shown to have an A1B specificity, unlike the A2 specificity of bee PLs. The complete amino acid sequences of the venom PL from the yellow jacket, Vespula maculifrons, and the more acidic isoenzyme from the white faced hornet, Dolichovespula maculata, have been determined by sequencing overlapping peptides isolated from enzyme and chemical digests of the proteins. Ves m 1 is composed of 300 amino acids, with variants found at 3 positions. Dol m 1.02 is composed of 303 amino acids with variants at 2 positions. Comparison with the sequence of Dol m 1.01 determined by cDNA sequencing gave 66.7% identity with Dol m 1.01 with almost all variation in the first 131 positions. Ves m 1 showed 69% identity with Dol m 1.01 and 38.7% with Dol m 1.02. Comparison of the PL sequences with the Protein Identification Resource data base showed many similarities wit h the lipase family, but very little relationship to known PLs. The amount of structural similarity between Dol m 1 and Ves m 1 is similar to that found among antigen 5 molecules of different genera, and is sufficient to account for antigenic cross-reactivity. There appear to be a number of highly conserved regions of the PL molecules, especially in the C-terminal 168 residues. These regions may from common epitopes found in several species and genera of vespids. Antibodies against these common epitopes will not be able to distinguish among PLs from various vespids.(

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[12] - Kolarich D, Léonard R, Hemmer W, Altmann F. The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris. FEBS J 2005;272:5182-5190

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.

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[13] - Hoffman DR. Hymenoptera venom allergens. Clin Rev Allergy Immunol 2006;30:109-128

Hymenoptera venoms each contain a variety of protein allergens. The major components have all been characterized, and most of the amino acid sequences are known. This article concentrates on the use of contemporary techniques including cloning, mass spectrometry and genomics in the characterization of venom allergens, and newer separation techniques for protein isolation. Examples of the use of these techniques with venom proteins are presented.

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[14] - Santos LD, Santos KS, de Souza BM, Arcuri HA, Cunha-Neto E, Castro FM, et al. Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae). Toxicon 2007;50:923-937

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing th at its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients

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[15] - Peiren N, de Graaf DC, Brunain M, Bridts CH, Ebo DG, Stevens WJ, et al. Molecular cloning and expression of icarapin, a novel IgE-binding bee venom protein. FEBS Lett 2006;580:4895-4899

The 1045bp full-length cDNA sequence of a new bee venom component was obtained by rapid amplification of cDNA ends. The 672bp coding sequence corresponds to a protein with a signal peptide and multiple carbohydrate binding sites, and it was named icarapin. It has the new consensus sequence N-[TS]-T-S-[TV]-x-K-[VI](2)-[DN]-G-H-x-V-x-I-N-[ED]-T-x-Y-x-[DHK]-x(2,6)- [STA]-[VLFI]-x-[KR]-V-R-[VLI]-[IV]-[DN]-V-x-P. At least two transcript variants were found. Recombinant icarapin was tested for recognition by IgE antibodies and gave a positive dot blot with sera from 4 out of 5 bee venom allergic patients, all beekeepers. Indirect immunofluorescent staining localized the protein in the cuticular lining of the venom duct

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[16] - Hoffman DR. Hymenoptera venom allergens. Clin Rev Allergy Immunol 2006;30:109-128

Hymenoptera venoms each contain a variety of protein allergens. The major components have all been characterized, and most of the amino acid sequences are known. This article concentrates on the use of contemporary techniques including cloning, mass spectrometry and genomics in the characterization of venom allergens, and newer separation techniques for protein isolation. Examples of the use of these techniques with venom proteins are presented.

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[17] - King TP, Guralnick M. Hymenoptera 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. 237-49

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[18] - Blank S, Seismann H, Bockisch B, Braren I, Bredehorst R, Ollert M, et al. Identification, recombinant expression and characterisation of high molecular weight hymenoptera venom allergens. Allergy 2008;63(suppl. 88):13-14

Background: Hymenoptera venoms are known to cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in allergic individuals. Although a panel of proteinic and peptidic compounds have been described as allergens so far, prominent compounds supposed to be major allergens still remain to be characterized. The aim of the present study was to identify and characterize the high molecular weight allergen Api m 5 (allergen C) in bee venom and its putative homologue in wasp venom. Methods: The respective proteins were enriched from the venoms of Apis mellifera and Vespula vulgaris and subjected to MS/ MS-based sequencing allowing the identification of a candidate gene for A. mellifera and V. vulgaris. Different PCR and 5‚RACE strategies were followed to amplify the coding sequences from venom gland cDNA and the obtained genes were cloned and expressed in Trichoplusia ni insect cells. The recombinant allergens were analyzed regarding their enzymatic activity and their immunoreactivity with serum IgE antibodies of hymenoptera venom-sensitised patients. Results: Data from MS/MS-based sequencing of the enriched allergens led to the identification of a candidate gene in the genomic database of A. mellifera although the 50-terminal segment differs significantly from the predicted gene. The precursor cDNA contains a 2328 bp open reading frame encoding a 775 amino acid protein. Since no genomic information for V. vulgaris is available so far homology-based approaches were applied and yielded the corresponding gene. The mature proteins comprise a dipetidyl peptidase IV domain and a prolyl oligopeptidase domain and exhibit a sequence identity of 53% and of 32% to the human enzyme (DPP IV). After expression in insect cells, enzymatic activity was assessed and compared to hymenoptera venom. Reactivity of a monoclonal human IgE antibody selected from a combinatorial library confirmed the presence of the identified protein in the venom. Additionally, the reactivity of the full length protein with sera of hymenoptera venom-sensitised patients proved the allergenic nature of the proteins. Conclusion: The identification of the first DPP IV enzymes in hymenoptera venoms and their recombinant availability may contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for both diagnostic and therapeutic approaches.

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[19] - Blank S, Seismann H, Bockisch B, Braren I, Bredehorst R, Grunwald T, et al. Identification and recombinant expression of a novel IgE-reactive 70 kDa carboxylesterase from Apis mellifera venom. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°30

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[20] - Peiren N, de Graaf DC, Brunain M, Bridts CH, Ebo DG, Stevens WJ, et al. Molecular cloning and expression of icarapin, a novel IgE-binding bee venom protein. FEBS Lett 2006;580:4895-4899

The 1045bp full-length cDNA sequence of a new bee venom component was obtained by rapid amplification of cDNA ends. The 672bp coding sequence corresponds to a protein with a signal peptide and multiple carbohydrate binding sites, and it was named icarapin. It has the new consensus sequence N-[TS]-T-S-[TV]-x-K-[VI](2)-[DN]-G-H-x-V-x-I-N-[ED]-T-x-Y-x-[DHK]-x(2,6)- [STA]-[VLFI]-x-[KR]-V-R-[VLI]-[IV]-[DN]-V-x-P. At least two transcript variants were found. Recombinant icarapin was tested for recognition by IgE antibodies and gave a positive dot blot with sera from 4 out of 5 bee venom allergic patients, all beekeepers. Indirect immunofluorescent staining localized the protein in the cuticular lining of the venom duct

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[21] - Soldatova LN, Crameri R, Gmachl M, Kemeny DM, Schmidt M, Weber M, et al. Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli. J Allergy Clin Immunol 1998;101:691-698

BACKGROUND: Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described. OBJECTIVE: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity. METHODS: In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells. RESULTS: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli. CONCLUSIONS: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya

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[22] - Grunwald T, Bockisch B, Spillner E, Ring J, Bredehorst R, Ollert MW. Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3). J Allergy Clin Immunol 2006;117:848-854

BACKGROUND: Acid phosphatase (Api m 3) is a major allergen in honeybee (Apis mellifera) venom, and its availability as a recombinant protein may facilitate the development of improved diagnostic tests and immunotherapies . OBJECTIVE: One objective is the determination of the complete primary structure of Api m 3 and to obtain recombinant Api m 3 on the basis of expression in insect cells. Another objective is the quantitative analysis of patient serum IgE antibody reactive to recombinant Api m 3 . METHODS: The cloning of Api m 3 from venom gland cDNA and its expression as a full-length protein in eukaryotic insect cells is described. The immunoreactivity of serum IgE antibodies of honeybee venom-sensitized patients to recombinant Api m 3 was determined in an enzyme immunoassay . RESULTS: PCR amplification generated a 1122-bp DNA fragment whose identity as the coding sequence of Api m 3 was verified by several means. Recombinant Api m 3, expressed in Trichoplusia ni cells, showed an expected molecular weight and enzymatic activity at pH 4.5. Analysis of tryptic fragments of purified recombinant Api m 3 by mass spectrometry confirmed its identity. In immunoassays, recombinant Api m 3 is specifically recognized by IgE antibodies of pooled serum in Western blots and by 37% of the individual sera of honeybee venom-sensitized patients in ELISA analysis . CONCLUSION: The availability of recombinant Api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy. CLINICAL IMPLICATIONS: The recombinant venom allergen Api m 3 is a key element in the search for an optimized component-resolved approach to honeybee venom allergy with regard to both the development of superior diagnostic tests and the improvement of allergen immunotherapy.

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[23] - Müller UR, Dudler T, Schneider T, Crameri R, Fischer H, Skrbic D et al. Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is similar. J Allergy Clin Immunol 1995;96:395-402

Phospholipase A2 is the major allergen in honeybee venom. Recombinant phospholipase A2 was produced in prokaryotes and tested for its biologic activity by intracutaneous skin testing with serial 10-fold dilutions in comparison with native and deglycosylated phospholipase A2 in patients allergic to bee venom. Linear regressions of the log of the wheal area versus the log of the allergen concentration were calculated for all allergens in each patient. The relative allergenic potency of the various preparations was analyzed by comparing the linear regressions. Native phospholipase A2 was about 10 times more potent than whole bee venom. None of 58 patients allergic to bee venom was missed by testing with native phospholipase A2 alone. This allergen and deglycosylated native phospholipase A2 resulted in similar skin reactions, indicating that the sugar residues were of little relevance for IgE-binding in the patients tested. Native phospholipase A2 also had relative potency similar to that of recombinant refolded phospholipase A2, whereas recombinant nonrefolded phospholipase A2 had almost no biologic activity in skin testing. These results demonstrate in vivo activity of the recombinant bee venom allergen phospholipase A2. Although correct refolding is a prerequisite for type I skin reactivity, glycosylation seems to be less important.

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[24] - Paull BR, Yunginger JW, Gleich GJ. Melittin: an allergen of honeybee venom. J Allergy Clin Immunol 1977;59:334-338

The presence of serum IgE antibodies to melittin was tested by the radioallergosorbent test (RAST). Melittin, the principal protein of honeybee venom, was isolated by gel filtration on Sephadex G-75 and covalently bound to cyanogen bromide-activated microcrystalline cellulose. The melittin preparation was homogenous by immunoelectrophoresis with the use of rabbit antiserum to whole honeybee venom and by polyacrylamide electrophoresis in gels containing 1% sodium dodecyl sulfate. Elevated serum IgE antibodies to melittin (three times greater than binding by normal sera) were found in 7 of 24 honeybee venom-sensitive persons and in 5 of 20 nonsensitive beekeepers. In one venom-sensitive patient a particularly high titer of IgE antibody was found. The reaction between solid-phase melittin and IgE antibody could be inhibited by fluid-phase melittin but not by phospholipase A (PLA). Similarly, the reaction of IgE antibody with solid-phase PLA was inhibited by PLA but not by melittin. In passive transfer skin tests with the sensitive patient's serum, positive wheal- and-flare reactions were obtained in 3 nonallergic recipients following melittin challenge; appropriate controls were negative. These results indicate that melittin is an allergen in some honeybee venom-sensitive patients and in an occasional patient melittin may be a major allergen

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[25] - Dudler T, Machado DC, Kolbe L, Annand RR, Rhodes N, Gelb MH, et al. A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2. J Immunol 1995;155:2605-2613

The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses

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[26] - Müller U, Fricker M, Wymann D, Blaser K, Crameri R. Increased specificity of diagnostic tests with recombinant major bee venom allergen phospholipase A2. Clin Exp Allergy 1997;27:915-920

BACKGROUND: In diagnosis of type I allergy recombinant allergens have potential advantages over conventional allergenic extracts, both regarding specificity and reproducibility. OBJECTIVES: We therefore decided to study honey bee venom (BV) and its major allergen phospholipase A2 (PLA) in native and recombinant form for diagnosis of bee sting allergy. METHOD: We investigated 85 patients with a history of a recent systemic allergic bee sting reaction and positive intracutaneous skin test to BV, and 21 controls with no history of allergic bee sting reaction and negative skin test to BV. Intracutaneous skin tests and determination of specific IgE by ImmunoCAP(R) to BV, native PLA (nPLA) and recombinant PLA (rPLA) were done in all patients and controls. RESULTS: In skin testing 84 (99%) of the 85 patients reacted to nPLA and 81 (95%) to rPLA, while none of the 21 controls was positive with nPLA or rPLA. Specific serum IgE to BV could be detected in 82 of the patients (96%), to nPLA in 73 (86%) and to rPLA in 66 ( 78%). Four (19%) of the controls had a positive CAP to BV, one (4.8%) to nPLA and none to rPLA. Analysis of discordant results in CAP showed, that most patients with specific IgE to BV, but not to nPLA and rPLA, had positive skin tests to both PLA preparations and low levels of BV specific IgE. Patients with specific IgE to nPLA but not to rPLA were usually sensitized to minor allergens of BV which contaminated the commercial nPLA. CONCLUSIONS: PLA is the major allergen in BV. While diagnostic tests with BV are more sensitive, the specificity of tests with PLA, especially rPLA is clearly increased as compared with BV

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[27] - Royer B, Harraga S, Meyer JP, Lustgarten V, Adessi B, Kantelip JP, et al. Valeur diagnostique des prick-tests, des IgE spécifiques et du test de libération d'histamine pour le diagnostic de routine d'allergie aux hyménoptères. Rev Fr Allergol Immunol Clin 2004;44:144-150

Aim: To study risk factors for lupine flour sensitization, especially in peanut-allergic children Patients: 515 consecutive children referred in an outpatient clinic for suspicion or follow-up of respiratory or food allergy, from November 2001 to November 2002 Methods: Prick-tests (PT) with commercially available lupine flour and peanut extracts (Allerbio Lab., Varennes-en-Argonne, France, and Stallergenes S.A., Antony, France, respectively). - comparison of demographic data and clinical history (CH) between 4 groups: children without sensitization to lupine flour and peanut, children sensitized to lupine flour or peanut only, and children sensitized to lupine flour and peanut. Case-control study of 17 peanut-allergic children (CH and positive PT) without lupine flour sensitization and 17 peanut-allergic children sensitized to lupine flour, matched for sex and age. RESULTS: 5 % of the children (26/515) were sensitized to lupine flour. Seventy-seven p. cent (20/26) and 65 % (17/26) of those children were sensitized or clinically allergic to peanut respectively, - based on CH and positive PT, 30 % (8/26) of the children with lupine flour sensitization were allergic to other legumes (peas, lentils, beans), independently of their sensitization to peanut, - 24 % (17/72) of the peanut-allergic children (CH or positive food challenge) were sensitized to lupine flour. Serum peanut IgE levels were significantly higher in children with peanut and lupine sensitization than in children with peanut sensitization only (p<0,001). CONCLUSION: Our results confirm the high frequency of cross-sensitizations between peanut and lupine flour. They show also that a CH of reactions to legumes such as peas, lentils or beans, is an important risk factor to lupine flour sensitization (OR = 16, CI = 4.78-54.69). The high levels of peanut-specific IgE in the serum of peanut-allergic children sensitized to lupine flour suggest that a sensitization to lupine flour might be a risk factor for persistence of peanut allergy. Furthermore, as sensitization to legumes can be a dynamic process, PT for lupine flour should be repeated in children with high or increasing serum peanut specific IgE levels.

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[28] - Mahler V, Gutgesell C, Valenta R, Fuchs T. Natural rubber latex and hymenoptera venoms share Immunoglobin E-epitopes accounting for cross-reactive carbohydrate determinants. Clin Exp allergy 2006;36:1446-1456

BACKGROUND: Epidemiological data on the prevalence and risk factors of latex sensitization have suggested a significant association between latex sensitization and the presence of one or more positive skin prick test responses to aeroallergens, food allergens and to one or more insect venoms. Xylose and core 3-fucose are typical complex glycans in plants and are foreign to mammals. Plant N-glycans and insect N-glycans may cross-react in humans . OBJECTIVE: The aim of our study was to investigate whether there are cross-reactive IgE-binding structures in natural rubber latex (NRL) and hymenoptera venoms and to examine their nature . METHODS: Hundred and twenty-five consecutive patients with insect venom allergy were screened for coincidental latex-specific IgE. IgE-binding components in the venoms from Apis mellifera and/or vespula species and in NRL extracts were characterized by IgE-immunoblotting to the natural allergen sources and determination of specific IgE to recombinant allergens. Cross-reactive components were investigated by inhibition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE-epitopes was further examined by specific IgE-binding to cross-reactive carbohydrate determinants (CCD) in bromelain and horseradish peroxidase as well as by periodate treatment . RESULTS: NRL glove extracts inhibited patients' serum IgE-binding to venom allergens. Vice versa, the IgE-binding to latex glove extracts could be inhibited by pre-incubation with the insect venoms. Specific IgE-binding to recombinant latex allergens was absent, whereas the cross-reactive IgE-epitopes were sensitive to periodate treatment and specific IgE to CCD (MMXF and MUXF type) could be detected . CONCLUSION: Insect venoms and NRL share IgE-binding CCD that may be responsible for positive serological test results to NRL in patients with insect venom allergy. This copositivity occurs frequently (13.6%) among venom-allergic individuals and did not elicit clinical symptoms upon contact to latex in the patients examined. In contrast, true cosensitization to insect venoms and NRL allergens can occur and may not be missed.

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[29] - Graif Y, Confino-Cohen R, Goldberg A. Reproducibility of skin testing and serum venom specific IgE in Hymenoptera venom allergy. Ann Allergy Asthma Immunol 2006;96:24-29

BACKGROUND: The decision regarding an immunotherapy regimen for venom-allergic patients is based on the results of skin testing and serum venom specific IgE measurements. However, their reliability has been questioned, and their reproducibility has not been examined. OBJECTIVE: To evaluate the reproducibility and reliability of the results of skin testing and serum venom specific IgE measurement in venom-allergic patients. METHODS: Patients with a systemic reaction after an insect sting were evaluated twice, 2 to 6 weeks apart, by intradermal skin tests and by determination of serum venom specific IgE to Hymenoptera venoms. RESULTS: Thirty-five patients were evaluated 1 to 168 months (mean, 23 months) after the sting reaction. Reproducibility of skin test results for all venoms at the 2 sessions was found in 23 patients (66%). Reproducibility of venom specific IgE results for all venoms was found in 16 (59%) of 27 patients from whom 2 blood samples were available for evaluation. Concordance between skin test and venom specific IgE results for all venoms was found in 30 (51%) of 59 samples available for evaluation. CONCLUSIONS: The reproducibility of venom skin test and serum venom specific IgE results is relatively poor. It is common practice for therapeutic decisions regarding venom immunotherapy to be based on a single diagnostic evaluation. Consequently, many patients are either overtreated or undertreated. Better diagnostic methods are required in venom allergy.

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[30] - Jappe U, Freising C. Hymenoptera venom allergy: Evaluation of in vitro- and in vivo-allergy diagnostic before, during and after specific immunotherapy. Allergy Clin Immunol Int 2005;17(Suppl. 1):183

Background. The diagnosis of stinging insect allergy is based upon standardized test procedures consisting of history, skin test and the detection of specific (s)IgE and IgG to the venoms of honey bee (HB) and yellow jacket (YJ). Most patients are protected after a specific immunotherapy (IT). However, in vivoand in vitro-diagnostic are not 100% specific and sensitive, and identification of the stinging insect sometimes impossible. The purpose of this study was to evaluate the diagnostic results of 582 patients with suspected hymenoptera venom allergy who had attended the allergy department of the University of Heidelberg, Germany, between November 1997 and January 2003. Methods. The following patients were further investigated: 1. those with an anaphylactic reaction and completed in vivo and in vitro-testing but who had not begun an IT yet (n=210); 2. patients under IT (n=51); and 3. patients after IT (n=25). The severity of the clinical reaction was graded applying the Mueller scale (I to IV) and sIgE and sIgG4 were detected using CAP-FEIA (Pharmacia Diagnostics, Freiburg, Germany) and Western Blot (DPC Biermann, Bad Nauheim, Germany). The blots were scanned and the intensity of the specific reactions determined semi-quantitatively with the software Quantiscan. Results. For 129 patients the results of the skin test did not correlate with the Mueller grade. In contrast, for n=196 there was a good correlation between Mueller grade and sIgE and sIgG4 (CAP-FEIA). Before IT, higher concentrations of sIgE and sIgG4 in Western Blot were correlated with the severity of the reaction (n=47). Patients allergic to YJ had sIgE mostly directed to antigen 5, phospholipase, and hyaluronidase. The correlation factor for the comparison between both in vitro-procedures was statistically significant with 0.691 for sIgE and 0.598 for sIgG4, the calculation based upon the respective highest peak. The comparison between skin test and CAP-FEIA revealed a highly significant correlation of sIgE and skin test, which was similar for sIgG4. Skin test and Western Blot showed a correlation for sIgE but not sIgG4. During IT sIgE initially increased in 56% of the patients and decreased shortly afterwards, whereas sIgG4 increased during the first up to the third year of IT and decreased after more than 2 years after IT discontinuation. Conclusion. The decrease of IgG4 years after IT could provide evidence for a decrease of the immunological as well as the protective effect of IT.

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[31] - Bilò BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JNG, et al. Diagnosis of hymenoptera venom allergy. Allergy 2005;60:1339-1349

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

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[32] - Ruëff F, Przybilla B, Müller U, Mosbech H. The sting challenge test in Hymenoptera venom allergy. Position paper of the Subcommittee on Insect Venom Allergy of the European Academy of Allergology and Clinical Immunology. Allergy 1996;51:216-225

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[33] - Hamilton RG. Diagnostic methods for insect sting allergy. Curr Opin Allergy Clin Immunol 2004;4:297-306

PURPOSE OF REVIEW: This review overviews advances from mid-2002 to the present in the validation and performance methods used in the diagnosis of Hymenoptera venom-induced immediate-type hypersensitivity. RECENT FINDINGS: The general diagnostic algorithm for insect sting allergy is initially discussed with an examination of the AAAAI's 2003 revised practice parameter guidelines. Changes as a result of a greater recognition of skin test negative systemic reactors include repeat analysis of all testing and acceptance of serology as a complementary diagnostic test to the skin test. Original data examining concordance of venom-specific IgE results produced by the second-generation Pharmacia CAP System with the Johns Hopkins University radioallergosorbent test are presented. Diagnostic performance of honeybee venom-specific IgE assays used in clinical laboratories in North America is discussed using data from the Diagnostic Allergy Proficiency Survey conducted by the College of American Pathologists. Validity of venom-specific IgE antibody in postmortem blood specimens is demonstrated. The utility of alternative in-vivo (provocation) and in-vitro (basophil-based) diagnostic testing methods is critiqued. SUMMARY: This overview supports the following conclusions. Improved practice parameter guidelines include serology and skin test as complementary in supporting a positive clinical history during the diagnostic process. Data are provided which support the analytical performance of commercially available venom-specific IgE antibody serology-based assays. Intentional sting challenge in-vivo provocation, in-vitro basophil flow cytometry (CD63, CD203c) based assays, and in-vitro basophil histamine and sulfidoleukotriene release assays have their utility in the study of difficult diagnostic cases, but their use will remain as supplementary, secondary diagnostic tests.

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[34] - Golden DBK. Insect sting allergy and venom immunotherapy: A model and a mystery. J Allergy Clin Immunol 2005;115:439-447

Whole-body extracts of Hymenoptera were used for diagnosis and treatment until controlled clinical trials proved them no better than placebo, whereas venom is 85% to 98% effective. Studies of natural history reveal why whole-body extracts were thought to work. The chance of future systemic reactions is low in large local reactors and in most children and varies between 20% and 70% in adults. Venom skin tests are most accurate, but RAST is an important complementary test. The degree of sensitivity on skin tests or RASTs does not reliably predict the severity of a sting reaction. Venom immunotherapy is recommended for patients at high risk for sting reactions. Rapid regimens are as safe as slower regimens. The recommended dose is 100 microg, but some patients require higher doses for full protection. Venom immunotherapy is continued every 4 to 8 weeks for at least 5 years in most cases. Skin test results become negative in only 25% after 5 years of therapy but in 60% to 70% after 7 to 10 years. When treatment is stopped after 5 years or more, there is a 10% chance of systemic reaction to each future sting, but most reactions are mild. Some patients have a higher risk of relapse and should continue treatment for an extended period.

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[35] - Ebo DG, Bridts CH, Hagendorens MM, de Clerck LS, Stevens WJ. The basophil activation test in the diagnosis and follow-up of hymenoptera venom allergy: an alternative point of view. J Investig Allergol Clin Immunol 2008;18:493-494

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[36] - Sainte-Laudy J, Sabbah A, Drouet M, Lauret MG, Loiry M. Diagnosis of venom allergy by flow cytometry. Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release. Clin Exp Allergy 2000;30:1166-1171

BACKGROUND: Potent allergens such as hymenoptera venoms are capable of inducing severe and life threatening clinical reactions. Percentage of false negative results obtained by the usual diagnostical methods is comprised between 10 and 25%. OBJECTIVE: Evaluation of the sensitivity and the specificity of cellular tests and particularly evaluation of a new flow cytometric method. METHODS: Forty-five allergic patients having experienced a local, a systemic reaction or an anaphylactic shock and 10 controls having undergone hymenoptera stings without clinical reactions were selected on the basis of the clinical history, skin tests and specific IgE. Three cellular tests were performed on the same cell suspensions and in the presence of 2 ng/mL of rIL3: histamine release (RIA), leukotriene C4 release (ELISA) and basophil activation test (flow cytometry after double anti-IgE FITC, anti-CD63 PE labelling). RESULTS: As compared to the clinical history, sensitivities of skin tests, specific IgE, flow cytometry, histamine release and leukotriene release were, respectively; 85%, 88%, 100%, 89% and 100%. Flow cytometric analysis of basophil activation showed a significant decrease of the mean fluorescence density and number of IgE positive cells and a significant increase of the number of CD63 positive cells. The 10 controls tested by flow cytometry were negative. CONCLUSION: As compared to the clinical history and to the other parameters tested here, flow cytometry showed a high sensitivity and a high specificity. The excellent correlation observed between this method and the other cellular tests such as histamine and leukotriene release are in favour of the specificity of flow cytomery and in favour of the use of this method for venom allergy diagnosis.

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[37] - Korosec P, Silar M, Erzen R, Bajroviæ N, Kosnik M. Basophil activation test is more sensitive than intradermal testing in patients with convincing histories of systemic reactions to a Hymenoptera sting, but negative other diagnostic tests. Allergy 2008;63(suppl. 88):82

Background: Current diagnostic guidelines do not adequately address the question how best to manage the patients with convincing history of insect allergy, but negative venom specific IgE and skin test results. Methods: Twenty-nine patients were recruited from the University Clinic of Respiratory and Allergic Diseases over a 3-year period. All patients had a convincing history of a severe or life-threatening anaphylactic reaction of Mueller grades II to IV (median grade IV) after a Hymenoptera sting, but negative venom-specific IgE and skin prick test results. Diagnostic work-up was prospectively followed up by intradermal skin testing and CD63 basophile activation test. We also included a control group of 25 subjects without a history of systemic reaction to Hymenoptera sting. Results: Nineteen of 29 (69%) patients had positive CD63 response after stimulation with bee and/or wasp venom. All these patients were positive with the cut-off of 15% of CD63 positive basophils. Thirteen of them had positive response to one venom (5 bee and 5 wasp venom) and 7 to both venoms. Only 8 of 20 patients (40%) had a positive intradermal skin test results. In control group only 2 of 25 (4%) subjects had positive basophile response and/or intradermal testing. Conclusion: We showed that in the complex cases with inconclusive diagnostic results, CD63 activation test could be a particularly useful and more sensitive then intradermal skin testing. Both test showed comparable high specificity.

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[38] - Erdmann SM, Sachs B, Kwiecien R, Moll-Slodowy S, Sauer I, Merk HF. The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy. Allergy 2004;59:1102-1109

BACKGROUND: As in vitro diagnosis of wasp venom sensitization by specific serum IgE has a sensitivity of only 60-80%, additional in vitro tests are desirable. Basophil activation is associated with the expression of CD63 and its measurement has been proposed as a novel in vitro test for immediate-type allergy. Furthermore, to date, no in vitro test exists to monitor successful specific immunotherapy (SIT) with wasp venom. Therefore, the potentially harmful sting challenge is still recommended . OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of wasp venom allergy with skin tests and measurement of specific IgE. Furthermore, we investigated whether BAT can predict the outcome of the sting challenge in patients on SIT . METHODS: Fifty patients with a systemic reaction caused by a wasp sting and 20 controls were studied. Intracutaneous tests were performed with wasp and bee venom in the suspected allergics. Specific IgE was determined by the CAP-FEIA method and basophil activation by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. Twenty-five patients were sting challenged 6 months after starting SIT and the BAT was repeated before challenge . RESULTS: Sensitivity of the intracutaneous tests, specific IgE and BAT was 100, 76, and 92%, respectively. Specificity of specific IgE and the BAT was 85 and 80%, respectively. The cut-off for a positive BAT was 15% CD63+ basophils. There was a positive correlation between IgE reactivity to wasp venom and the number of CD63+ basophils (r = 0.65). Although no patient had a systemic reaction upon sting challenge, in most subjects basophil activation did not decrease when compared with the BAT before SIT . CONCLUSIONS: Quantitation of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of allergy to hymenopteran venoms. The CD63-based BAT is a helpful tool for the complementation of routine diagnostic tests such as specific IgE as it increases sensitivity of in vitro detection of sensitization. However, this in vitro method does not offer an alternative to the sting challenge in monitoring successful SIT.

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[39] - Sturm GJ, Böhm E, Trummer M, Weiglhofer I, Heinemann A, Aberer W. The CD63 basophil activation test in Hymenoptera venom allergy: a prospective study. Allergy 2004;59:1110-1117

BACKGROUND: The basophil activation test (BAT), which relies on flow cytometric quantitation of the allergen-induced up-regulation of the granule-associated marker CD63 in peripheral blood basophils, has been suggested to be a useful approach in detecting responsiveness to allergens. The purpose of this study was to establish the usefulness of the BAT with regard to the clinical history and current diagnostic tools in Hymenoptera venom allergy using a prospective study design . METHODS: Fifty-seven consecutive patients allergic to Hymenoptera venom as defined by a systemic reaction after an insect sting, and 30 age- and sex-matched control subjects with a negative history were included. The degree and nature of sensitization was confirmed by skin testing, specific immunoglobulin E (IgE), serum tryptase levels and BAT. In the nonallergic control group only analysis of specific IgE and BAT were performed. Correlation of BAT, skin test and specific IgE, respectively, with the clinical history in the allergic group was termed as sensitivity and in the control group as specificity . RESULTS: Twenty one of 23 (91.3%) bee venom allergic patients and 29 of 34 (85.3%) patients allergic to wasp and hornet venom tested positive in BAT. The overall sensitivity of BAT, specific IgE and skin tests were 87.7, 91.2 and 93.0%, respectively. The overall specificities were 86.7% for BAT and 66.7% for specific IgE. No correlation between the severity of clinical symptoms and the magnitude of basophil activation was observed . CONCLUSION: The BAT seems to be an appropriate method to identify patients allergic to bee or wasp venom with a comparable sensitivity to standard diagnostic regimens. The higher specificity of BAT as compared with specific IgE makes this test a useful tool in the diagnosis of Hymenoptera venom allergy.

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[40] - Bilò BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JNG, et al. Diagnosis of hymenoptera venom allergy. Allergy 2005;60:1339-1349

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

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[41] - Müller U, Golden DB, Lockey RF, Shin B. Immunotherapy for hymenoptera venom hypersensitivity. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 377-92

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[42] - Bilò BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JNG, et al. Diagnosis of hymenoptera venom allergy. Allergy 2005;60:1339-1349

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

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[43] - Sturm GJ, Schuster C, Kranzelbinder B, Wiednig M, Groselj-Strele A, Aberer W. Asymptomatic Sensitization to Hymenoptera Venom Is Related to Total Immunoglobulin E Levels. Int Arch Allergy Immunol 2009;148:261-264

BACKGROUND: The detection of specific serum immunoglobulin E (sIgE) to Hymenoptera venoms is an established diagnostic tool to diagnose insect venom hypersensitivity. However, the specificity of sIgE detection is a debated issue . METHODS: In 145 subjects, total IgE (tIgE) and sIgE to Hymenoptera venoms as well as sIgE to rapeseed as a marker of cross-reactive carbohydrate determinants were measured. In addition, an atopy score was determined for each patient. We looked for a possible association between tIgE and the presence of sIgE in subjects with a negative history of large local or systemic reactions to insect stings . RESULTS: Fifteen of 65 subjects (23.1%) with low levels of tIgE (<50 kU/l) had sIgE for bee or wasp venom, and 23 of 47 subjects (48.9%) with a tIgE from 50 to 250 kU/l showed sIgE. The highest rate of asymptomatic sensitization (22 of 33; 66.7%) was found in patients with tIgE levels higher than 250 kU/l. Median sIgE was approximately 4.8 times higher in subjects with tIgE levels above 250 kU/l than in those with tIgE levels <50 kU/l. Interestingly, a significant difference in median tIgE was recorded between individuals with and without sIgE to rapeseed [776.5 kU/l (25, 75% percentiles: 252.5, 2,000.0) vs. 50.5 kU/l (20.1, 172.0), respectively; p < 0001] . CONCLUSION: Specific antibodies are frequently seen in individuals with high tIgE, but appear to be largely irrelevant in clinical terms. This might lead to misdiagnosis in persons with an inconclusive sting history.

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[44] - Golden DBK, Tracy JM, Freeman TM, Hoffman DR. Negative venom skin test results in patients with histories of systemic reaction to a sting. J Allergy Clin Immunol 2003;112:495-498

For more than 20 years venom immunotherapy has been the preferred treatment for Hymenoptera allergy and venom skin testing the preferred diagnostic test. Most allergists consider venom skin tests to be highly accurate and interpret a negative venom skin test result to indicate the absence of insect allergy. Furthermore, current practice guidelines do not adequately address the question of how best to manage the patient with a convincing history of insect allergy but negative skin test results. Recent case reports and published studies have forced us to reexamine this important management issue and to consider what role in vitro venom testing might have in the management of insect allergy. We reviewed the current status of what is known about the management of individuals with a history of insect allergy but negative venom skin test results and suggested modifications of current working guidelines.

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[45] - Monsalve R, Gutiérrez R, Polo F, Lombardero M, Galán A, King T, et al. Use of purified venom allergens in the ADVIA-Centaur® platform for improving the diagnosis of vespid venom allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°326

Background:Recombinant antigen 5s, rVes v 5 and rPol a 5, had been used as specific reagents in the ADVIA-Centaur® platform and have allowed to measure specific IgEs in about 70% of allergic patients, also permitting in most of them to discriminate on their primary sensitivity towards Vespula or Polistes species. Nevertheless, in order to increase this level of detection and therefore obtain a more complete diagnosis of allergic patients, other vespid venom allergens were used as specific reagents of this IgE-measuring platform. Methods:Purified major venom allergens from Vespula vulgaris (Ves v 1, Ves v 2 and Ves v 5) and Polistes dominulus (Pol d 1 and Pol d 5), as well as bee venom phospholipase (Api m 1) have been biotinylated and individually used as specific reagents in the ADVIA-Centaur® platform, in order to measure specific IgE in the sera of patients allergic to wasps stings. Results:In most of the cases (>90% of patients tested) specific IgE can be measured by using isolated vespid venom allergens as specific reagents in the ADVIA-Centaur® platform. The relative importance of A1 phospholipases (Ves v 1 and Pol d 1) and antigen 5s (Ves v 1 and Pol d 5) is shown, demonstrating their importance as allergenic components of the venom. This response is variable among different patients, contributing to differentiate between their sensitivity to Vespula or Polistes. Conclusions:The availability of purified venom allergens from Vespula and Polistes species allows a more precise diagnosis of patients allergic to vespid stings.

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[46] - Müller UR, Johansen N, Petersen AB, Fromberg-Nielsen J, Haeberli G. Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m 1 and Ves v 5. Allergy 2009;64:543-548

BACKGROUND: In patients with hymenoptera venom allergy diagnostic tests are often positive with honey bee and Vespula venom causing problems in selection of venoms for immunotherapy . METHODS: 100 patients each with allergic reactions to Vespula or honey bee stings and positive i.e. skin tests to the respective venom, were analysed for serum IgE to bee venom, Vespula venom and crossreacting carbohydrate determinants (CCDs) by UNICAP (CAP) and ADVIA Centaur (ADVIA). IgE-antibodies to species specific recombinant major allergens (SSMA) Api m1 for bee venom and Ves v5 for Vespula venom, were determined by ADVIA. 30 history and skin test negative patients served as controls . RESULTS: By CAP sensitivity was 1.0 for bee and 0.91 for Vespula venom, by ADVIA 0.99 for bee and 0.91 for Vespula venom. None of the controls were positive with either test. Double positivity was observed in 59% of allergic patients by CAP, in 32% by ADVIA. slgE to Api m1 was detected in 97% of bee and 17% of Vespula venom allergic patients, slgE to Ves v5 in 87% of Vespula and 17% of bee venom allergic patients. slgE to CCDs were present in 37% of all allergic patients and in 56% of those with double positivity and were more frequent in bee than in Vespula venom allergic patients . CONCLUSIONS: Double positivity of IgE to bee and Vespula venom is often caused by crossreactions, especially to CCDs. IgE to both Api m1 and Ves v5 indicates true double sensitization and immunotherapy with both venoms.

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[47] - Müller U, Golden DB, Lockey RF, Shin B. Immunotherapy for hymenoptera venom hypersensitivity. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 377-92

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[48] - Caruso B, Bonadonna P, Severino MG, Manfredi M, Dama A, Schiappoli M, et al. Evaluation of the IgE cross-reactions among vespid venoms. Allergy 2007;62:561-564

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[49] - Bilo M, Brianzoni M, Cinti B, Frontini F, Bonifazi F. Diagnosis of polistes venom allergy in Europe. Allergy 2008;63(suppl. 88):442

Background: Although American Polistes (AP) species are not present in Europe and a weaker cross-reactivity between European and American Polistes was recently demonstrated, AP mixtures are usual for diagnosis and therapy. Aim: To compare cutaneous and serological reactivity using Polistes dominulus (Pd) versus AP venoms. Methods: We studied 33 Italian patients with a history of systemic reactions to Vespid (yellow jacket and/or paper wasp) stings. Stallergenes (Milan, Italy) and Alk- Abello` (Milan, Italy) supplied Apis mellifera, Vespula spp. and AP venoms for skin testing. The Pd venom was purchased from Anallergo (Florence, Italy). Serum specific IgE antibodies were detected using the CAPsystem (Phadia, Uppsala, Sweden). Results: All patients were skin positive to Polistes genus. In 18 (55%) cases the paper wasp was identified as the culprit insect. Thirteen patients (39%) were monosensitive to the genus Polistes by skin test, of which five (38%) were positive to Pd alone; among them, 4 had a history of a grade IV and 1 a grade III Mueller reaction. Of the remaining polysensitive patients (20/33), one individual who recognised the culprit insect, tested positive to an intradermal skin test for Pd venom alone. Out of 28 patients positive to both AP and Pd venoms, skin reactivity to the Pd was greater in 16 (57%). In 12 patients (43%) skin reactivity was identical.- Out of all patients, AP in 77% of cases and Pd in 90% yielded positive CAP-system results. In AP and Pd species CAP-positive patients, the trend was towards higher IgE titres to the Pd species. Specific IgE to AP alone was never detected. In the 5 Pd alone positive skin tests, specific IgE antibodies to AP were negative, while in 3 of them the CAP-system detection was positive to Pd. Conclusion: Skin reactivity to Pd extract was greater than that to the AP in more than 50% of cases. Skin tests were positive to Pd venom alone in 18% of patients with a history of severe systemic reactions. Pd venom yielded positive CAP-system results in more cases than did AP. These findings clearly suggest the need to replace the AP mix extract with an European Polistes extract for both diagnostic and therapeutic purposes. Indeed, omitting to perform skin tests for Pd extract would lead to the wrong diagnosis of non IgE-mediated anaphylaxis and consequently deprive the patients of life-saving immunotherapy.

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[50] - Severino MG, Campi P, Macchia D, Manfredi M, Turillazzi S, Spadolini I et al. European Polistes venom allergy. Allergy 2006;61:860-863

The American Polistes species venom mixture--that of P. annularis, P. fuscatus, P. metricus and P. exclamans--was the only commercially available mixture for diagnosis and therapy until 1996. However, these species of Polistes are not present in Europe, where P. dominulus and P. gallicus and to a lesser extent P. nimphus are widespread. The aim of this study was to assess the allergenic differences among the commercial American mix, P. dominulus and P. gallicus venom in European patients and therefore to verify if this mixture is suitable for diagnosis in these patients. We carried out skin tests, radioallergosorbent tests (RAST) and RAST inhibition in Italian patients with adverse reactions to Polistes stings. RAST inhibition results demonstrated that cross-reactivity between the American and European species is only partial and that P. dominulus and P. gallicus venoms have exclusive allergens. Skin tests and direct RAST confirmed these results and also showed that European Polistes venom is more suitable than the American mix in Italian patients. Moreover, we found a high rate of cross-reactivity between P. dominulus and P. gallicus. To conclude, P. dominulus and/or P. gallicus venoms are necessary for diagnosis and therefore in the therapy of European patients.

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[51] - Bonadonna P, Caruso B, Labardi D, Dama A, Senna G, Passalacqua G. Treatment with American Polistes venom was ineffective in an Italian patient allergic to European Polistes. Allergy 2007;62:966-967

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[52] - Severino M, Caruso B, Bonadonna P, Macchia D, Campi P, Dama A, et al. Cross reactivity between European hornet and yellow jacket venoms. Allergy 2008;63(suppl. 88):79

Background: The choice of the vaccine for immunotherapy (IT) is crucial in hymenoptera venom allergy (HVA). European Hornet (Vespa crabro) is largely present in many European countries and is now recognized as an important cause of severe reactions in patients with HVA. We evaluated the presence and extent of cross-reactivity between the venoms of Vespa crabro and Vespula germanica (Yellow Jacket) in patients with severe reactions to Vespa crabro stings. The cross reactivity was evaluated with CAP-inhibition techniques. Methods: Sera from patients with severe reactions (grade III and IV), and who unequivocally recognized Vespa crabro as the stinging insects were collected for the CAP-inhibition experiments. All patients underwent the standard diagnostic workup, including clinical history, skin prick test, intradermal tests and specific IgE measurement. The CAP inhibition test was carried out with a specific program in UniCap100 (Phadia, Uppsala, Sweden). The extent of homologous (blockage of venom-specific IgE by the same venom) and heterologous (blockage of the venom-specific IgE by the other venom) inhibition was computed with the following formula: %inhibition=100- [IgE inhibited sample (kU/L)x100/IgE antivenom (kU/L) at zero concentration]. An inhibition higher than 75% was considered indicative of cross-reactivity. Results: Seventeen patients (12 male, mean age 45.3 years) had a severe reaction (10 grade IV and 7 grade III) and unequivocally identified the responsible insect as Vespa crabro. All had skin and CAP positivity to both Vespa crabro and Vespula germanica, with specific IgE of 3.98±2.55 and 10.2±19.6, respectively. In 11/17 patients, vespula venom efficiently bound the Vespa Crabro specific IgE, whereas the opposite did not occur. In 6 subjects the CAP-inhibition test provided inconclusive results, therefore hornet venom was used for vaccination, based on the identification of the stinging insect. Conclusion: In our experience, the positivity to both yellow jacket and European hornet, often makes difficult the choice of the vaccine. The CAP-inhibition assay, indeed evidenced that the two venoms extensively cross-react in 67% of patients. It can be hypothesized that the majority of patients are primarily sensitised to yellow-jacket, and the cross-reactivity is responsible for the severe reactions to hornet.

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[53] - Stapel SO, Waanders-Lijster de Raadt J, van Toorenenbergen AW, de Groot H. Allergy to bumblebee venom. II. IgE cross-reactivity between bumblebee and honeybee venom. Allergy 1998;53:769-777

To obtain more information on IgE cross-reactivity between bumblebee venom and honeybee venom, we tested sera from venom-sensitized patients for specific IgE against venoms from the European bumblebee (Bombus terrestris), the North American bumblebee (Megabombus pennsylvanicus), and the honeybee (Apis mellifera). RAST, RAST-inhibition, and immunoblotting experiments indicate that bumblebee venom and honeybee venom contain venom-specific IgE-binding epitopes. These results suggest that immunotherapy using honeybee venom may not be effective in all bumblebee venom-allergic patients. Our experiments also revealed differences in IgE binding for venom from European and American bumblebees.

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[54] - Lavaud F, Fontaine JF, Deslée G, Sabouraud D, Lebargy F. Peut-on arrêter une désensibilisation aux venins d'hyménoptères ? Rev Fr Allergol Immunol Clin 2004;44:276-280

Les indications de mise en route de l'immunothérapie spécifique aux venins d'hyménoptères sont bien codifiées après un bilan diagnostique lui aussi correctement défini. Le traitement entrepris, la question de sa durée va rapidement se poser au médecin et au patient. Les différentes études publiées sur le suivi et le devenir des patients après l'arrêt du traitement montrent que le risque de voir réapparaître une réaction systémique n'est pas nul et se chiffre en moyenne autour de 5 à 10 %. Ces réactions sont habituellement modérées et surviennent chez les sujets à risque. Plus que des paramètres diagnostiques tels que l'évolution des tests cutanés, des dosages d'IgE et d'IgG sériques spécifiques, ou les tests de provocation, c'est la durée du traitement qui peut être actuellement considérée comme critère d'arrêt. Ainsi une durée minimale de cinq ans est habituellement conseillée. Sur des terrains à risque (mastocytose, réactions anaphylactiques inaugurales sévères, réactions systémiques lors des injections de rappel), le traitement doit être prolongé éventuellement à vie. En tout état de cause, si l'arrêt du traitement est décidé, ce doit être avec le plein accord d'un patient éclairé sur les risques éventuels et disposant d'une trousse d'urgence comprenant de l'adrénaline auto-injectable.

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[55] - Tavares B, Loureiro G, Pereira C, Faria E, Cunha R, Chieira C. Dynamics of specific IgE to Apis mellifera and Vespula spp. venom in patients submitted to specific immunotherapy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°377

Background: Specific immunotherapy frequently lead to a decrease in specific IgE levels after the first year of treatment. In patients allergic to Hymenoptera venom it is desirable that specific IgE levels become indoseable after treatment. The purpose of this study was to evaluate the dynamics of specific IgE in patients submitted to Apis mellifera venom specific immunotherapy. METHODS: The authors assessed 23 patients allergic to honeybee venom (mean age 36±14.2 years of age; 6 female and 17 male) for specific Apis mellifera and Vespula spp. venom IgE levels before treatment with A. mellifera venom and in the first, third and fifth years of treatment (UniCap˙, Pharmacia). Statitics were based on Wilcoxon signed ranks test for paired samples; p<0.05 was considered statisticaly significant. RESULTS: The majority of patients (12 - 52.2%) had class III (Müller) anaphylactic reaction, 9 patients (39.1%) had class IV and 3 patients (8,6%) class II. 13 patients were also sensitized to Vespula spp. venom. 47.8% of patients were sensitized to at least one common aeroalergen. The analysis of the mean value of Apis mellifera and Vespula spp. venom specific IgE levels before and after the treatment revealed a greater decrease in the fifth year of treatment. These diferencies were statisticaly significant in the third and fifth years of treatment (Table 1). Before treatment First year of treatment Third year of treatment Fifth year of treatment IgE to Apis mellifera (KU/l) 19.69±24.25 14.29±16.68 6.26±7.874 5.81±7.69 p value 0.08 0.001 0 IgE to Vespula spp. (KU/l) 2.15±2.73 1.52±1.63 0.70±1.19 0.66±1.04 p value 0.093 0.008 0.009 Only two patients achieved zero values for specific IgE to A. mellifera in the fifth year of treatment. CONCLUSION: Specific immunotherapy had a positive effect in the reduction of specific IgE levels for both venoms. Nevertheless, this effect can not be assumed as an indicator of treatment efficacy.

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[56] - Haye R, Døsen LK. Insect sting allergy. A study from 1980 to 2003 of patients who started treatment with venom immunotherapy between 1980 and 1998. Clin Mol Allergy 2005;3:12

BACKGROUND: Previously we treated patients with insect sting allergy with venom immunotherapy (IT) using whole body insect extracts. From 1980 we changed to insect venoms. The purpose of this study was to analyse data from the patients in order to improve our treatment . METHODS: This is an open, single centre study on patients treated with venom IT 14 years or older with a history of a systemic allergic reaction to an insect sting, a positive skin prick test (SPT) or a positive RAST and willingness to comply with five years of IT. Clinical and laboratory data were registered prospectively at the start of IT and after five years of treatment until 2003 on patients who started IT between 1980 and 1998. Questionnaires were answered in 1989, 1993 and 2003. Statistical analysis was done with Pearson's chi square, Fisher's exact or the t-test . RESULTS: Of 315 patients treated, 44 were given bee, 248 common wasp and 23 both venoms. Of the common wasp sting incidents 5.5 % resulted in a severe allergic reaction (SAR) during adequate IT and 22% after cessation. Seventy-one per cent of the patients carried epinephrine. Precautionary steps were taken by 77% of the patients during or after inadequate IT. On or after adequate IT 83% felt completely or substantially safe. Surprisingly 29 % of those inadequately treated felt safer and 50% were satisfied with having had the opportunity to be treated. The SPT became negative in 68% of the wasp allergic patients after five years of adequate IT. Increased risk of experiencing SAR to a future sting in wasp allergic patients after cessation of adequate IT was significantly associated with a SAR due to IT during the rush regimen. SAR due to IT occurred very rarely during maintenance dosing . CONCLUSION: Adequate venom IT is very effective while ongoing but somewhat less effective after cessation, while inadequate treatment gives poor results. More of our patients should complete five years of IT and some should continue IT. The type of reaction to IT during incremental dosing may be of help in deciding who should continue beyond five years. Maintenance IT may be taken over by the general physician.

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[57] - Pravettoni V, Primavesi L, Bertolotti F, Piantanida M, Pastorello E. Venom immunotherapy and specific venom IgE: how do their levels decrease in stung and not stung patients? Allergy 2008;63(suppl. 88):436

Background: Specific Venom Immunotherapy (VIT) is a lifesaving treatment because patients undergoing VIT generally well tolerate field stings. Usually at the end of a 5-year long VIT a decrease in specific serum IgE and skin tests reactivity is observed, being a complete negativity rare. No clear IgE decrease cut-off has been associated with VIT efficacy. In this study we evaluated the percentage of specific venom IgE decrease in three populations: not stung patients, stung in the firs three yrs or in the last two yrs of VIT. Methods: We retrospectively evaluated 167 patients submitted to 5-year long VIT, 137 allergic to Yellow Jacket, YJ (85 male and 52 female, mean age 43) and 30 to Honey Bee, HB (14 male and 6 female, mean age 43). Specific venom IgE levels were determined at the baseline, after 3 and 5 yrs of VIT. Skin tests (prick at 100 mcg/mL and intradermal at 0.1 and 1 mcg/mL) were performed at the same time. Results: Out of 167 YJ allergic patients, 72 were stung during VIT (39 within the first three yrs and 28 in the last two). In not stung patients, at the 3 yr control, the specific IgE decrease was 72.7% (P<0.01) and, at the end, 82.9% (P<0.001). In the group of patients stung during the first 3 yrs no statistically significant decrease was observed (38.3%). These patients, anyway, at the 5 yr control, reached a percentage decrease of 71.7 (P<0.05) in respect to the baseline level. Patients stung during the last two yrs of VIT, at the 3 yr control, showed a similar decrease of not stung patients (66.3%, P<0.001); while at the end the decrease from baseline was 72.4%. Out of 30 HB allergic patients, 16 were beekeepers and were stung continuously during VIT. Not stung patients presented, at the intermediate control, a mean IgE decrease of 76.5% (P< 0.05) and finally of 88.5% (P<0.05). Stung patients showed a 35.7 and 60.1% at 3 and 5 yr control, respectively, no statistically significant decrease was found during VIT. Conclusion: Not stung patients showed a considerable mean IgE decrease during VIT both in YJ (83%) and HB (88%). On the contrary, re-stung patients, clinically protected, showed a lower IgE decrease from the baseline, in some cases not statistically significant. As the persistence of elevated specific IgE levels is one of the criteria for not discontinuing VIT, these results highlight that in stung patients elevated IgE level at the end of VIT are not surprising or alarming, particularly in HB allergic patients.

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[58] - Moneret-Vautrin M. Arrêt d'une immunothérapie spécifique aux venins d'hyménoptères. Alim'Inter 2008;13:142-144

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[59] - Ebo DG, Bridts CH, Hagendorens MM, de Clerck LS, Stevens WJ. The basophil activation test in the diagnosis and follow-up of hymenoptera venom allergy: an alternative point of view. J Investig Allergol Clin Immunol 2008;18:493-494

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[60] - Simons FER, Frew AJ, Ansotegui IJ, Bochner BS, Golden DBK, Finkelman FD, et al. Risk assessment in anaphylaxis: Current and future approaches. J Allergy Clin Immunol 2007;120(1 suppl.):S2-S24

Risk assessment of individuals with anaphylaxis is currently hampered by lack of (1) an optimal and readily available laboratory test to confirm the clinical diagnosis of an anaphylaxis episode and (2) an optimal method of distinguishing allergen-sensitized individuals who are clinically tolerant from those at risk for anaphylaxis episodes after exposure to the relevant allergen. Our objectives were to review the effector mechanisms involved in the pathophysiology of anaphylaxis; to explore the possibility of developing an optimal laboratory test to confirm the diagnosis of an anaphylaxis episode, and the possibility of improving methods to distinguish allergen sensitization from clinical reactivity; and to develop a research agenda for risk assessment in anaphylaxis. Researchers from the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergology and Clinical Immunology held a PRACTALL (Practical Allergy) meeting to discuss these objectives. New approaches being investigated to support the clinical diagnosis of anaphylaxis include serial measurements of total tryptase in serum during an anaphylaxis episode, and measurement of baseline total tryptase levels after the episode. Greater availability of the test for mature beta-tryptase, a more specific mast cell activation marker for anaphylaxis than total tryptase, is needed. Measurement of chymase, mast cell carboxypeptidase A3, platelet-activating factor, and other mast cell products may prove to be useful. Consideration should be given to measuring a panel of mediators from mast cells and basophils. New approaches being investigated to help distinguish sensitized individuals at minimum or no risk from those at increased risk of developing anaphylaxis include measurement of the ratio of allergen-specific IgE to total IgE, determination of IgE directed at specific allergenic epitopes, measurement of basophil activation markers by using flow cytometry, and assessment of allergen-specific cytokine responses. Algorithms have been developed for risk assessment of individuals with anaphylaxis, along with a research agenda for studies that could lead to an improved ability to confirm the clinical diagnosis of anaphylaxis and to identify allergen-sensitized individuals who are at increased risk of anaphylaxis.

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[61] - Hulikova K, Kucera P, Cvackova M, Wolfova E. Basophil reactivity (CD63 expression) in patients with Hymenoptera venom allergy. Allergy 2008;63(suppl. 88):82

Background: Basophil activation test (BAT), which is based on flow cytometric quantification of allergen-induced CD63 upregulation on peripheral blood basophils, has been proposed for in vitro diagnosis of IgEmediated reaction. The usefulness of BAT in prognosis of clinical symptom severity and monitoring of allergen immunotherapy efficacy is not completely elucidated. The purpose of this study was to investigate the relationship between basophil activation in patients with Hymenoptera allergy and reaction severity and duration of venom immunotherapy (VIT). Methods: Thirty patients (age range 24-72 years, mean age 52) with insect venom anaphylaxis were selected for the study on the basis of clinical history, skin prick tests and serum level of specific IgE. Patients underwent treatment with standardized aluminium based allergen extract. Five healthy subjects without clinical reactions were enrolled as negative controls. BAT using CD63 expression on CD203c-identified basophils was performed after stimulation of blood samples with different concentrations of bee and wasp venom (0.01-1 mg/mL). The data were expressed as the percentage of CD631cells. Linear regression and t-test were used for statistical evaluation. Results: In the group of patients with the most severe reaction (grade IV) the mean proportion of CD631cells at the highest allergen concentration was 30%, SD 20.95, compared to 31.67%, SD 21.34 (P=0.8331) in the group of patients with less severe reaction (grade I to III). At intermediate allergen concentration, the mean percentage of CD631cells was 17.3, SD 20.32 and 21.34, SD 17 (P=0.5634). Basophil activation at the lowest venom concentration also did not significantly differ in both groups. The range of VIT duration (maintenance dose) was 1-60 months (mean 23.55, SD 18.01). We did not find any significant correlation between VIT duration and basophil activation (r=-0.0363, 95% CI -0.391 to 0.328, P=0.849). Conclusion: In our group of patients, basophil activation correlated neither with reaction severity nor VIT duration. This lack of relationship can be possibly caused by basophil response heterogeneity. The question if the induction phase of VIT is more responsible for basophil tolerance induction than maintenance phase remains unresolved.

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[62] - Peternelj A, Silar M, Erzen R, Kosnik M, Korosec P. Correlation between basophil specific sensitivity and efficiacy of venom immunotherapy. Allergy 2008;63(suppl. 88):82

Background: Treatment failure of venom immunotherapy (VIT) is not rare and the risk and pathogenic factors for those failures are so far poorly understood. For that reason we evaluated allergen-specific basophil sensitivity in patients, who did not tolerate field re-stings after completed VIT treatment. Methods: Basophil responsiveness was evaluated by flow cytometry analyses of basophil CD63 surface expression induced by different concentrations of bee or wasp venom (1, 0.1 and 0.01 mg/mL) in 14 treated patients who had experienced systemic allergic reactions (Muller grades II-III) and 17 treated patients who had no reactions after the field re-stings. We also included a group of 28 Hymenoptera venom allergic patients who have not received VIT. Results: In 14 patients who still reacted to bee or wasp sting, basophil response at a venom concentration of 0.1 mg/mL was significantly higher then in patients who tolerated field re-stings (P=0.03; t-test). Basophil response was also slightly higher at a concentration of 1 mg/mL, but not to statistical significance (P=0.12; t-test). There was no difference in the response to direct cross-linking of the IgE and in venom specific IgE and IgG4 serum concentrations between those two groups (P>0.8; Fischer exact test, t-test). Patients who tolerated field re-stings have also significantly lower basophil response in comparison to patients who have not received VIT, both at 0.1 and 1 mg/mL of venom concentrations (P< 0.001; t-test). Conclusions: The results suggest that basophil venom specific sensitivity is associated with the efficiency of venom immunotherapy.

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[63] - Müller U, Golden DB, Lockey RF, Shin B. Immunotherapy for hymenoptera venom hypersensitivity. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 377-92

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[64] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[65] - Hemmer W, Focke M, Jarisch R. In vitro double positivity to bee and wasp venom: specific role of IgE antibodies against cross-reactive carbohydrate structures in Hymenoptera venom glycoproteins. Allergologie 1999;22(suppl. 9):S63-S64

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[66] - Hemmer W, Focke M, Kolarich D, Dalik I, Götz M, Jarisch R. Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N-glycans as cross-reactive allergens in honeybee and yellow jacket venom. Clin Exp Allergy 2004;34:460-469

BACKGROUND: IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging-insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy . OBJECTIVE: To confirm the role of carbohydrate epitopes in double positivity and to locate the responsible glycoallergens in HB and yellow jacket (YJ) venom by western blot . METHODS: Immunoblot inhibition using HB venom, YJ venom and two glycoprotein sources displaying 1-3-fucosylated N-glycans (i.e. oilseed rape (OSR) pollen, and the synthetic neo-glycoprotein fucosylated/xylosylated N-glycans from bromelain coupled to bovine serum albumin (MUXF-BSA)) as inhibitors were performed with sera from 15 double-positive patients with stinging-insect allergy. Additionally, reactivity with blotted hymenoptera venoms of a carbohydrate-specific rabbit antiserum against OSR pollen was investigated . RESULTS: Major venom glycoallergens binding with carbohydrate-specific human IgE and rabbit IgG were detected in HB venom at 42 (hyaluronidase (HYA)), 46, 65 and 95 kDa, and in YJ venom at 38 and 43 kDa (HYA). Antibody binding to these allergens was completely lost after periodate treatment. Glycans of HB phospholipase were bound by patients' IgE only after protein denaturation. In 10 of the 15 patients the reactivity was with the second venom because of carbohydrates alone. The high-molecular-weight glycoallergens identified in HB venom probably correspond to similar proteins described earlier, including allergens B and C. The 38-kDa YJ allergen might represent a homologue of V mac 3 . CONCLUSIONS: The data confirm the proposed role of carbohydrate-specific IgE in double positivity to HB and YJ venom and shed new light on some previously described minor hymenoptera allergens of uncertain clinical significance. The consideration of carbohydrate-specific IgE may allow to discriminate between patients with potentially relevant and patients with non-relevant double sensitization.

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[67] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[68] - Hemmer W, Jin C, Focke M, Jarisch R, Altmann F. Yellow Jacket Venom Hyaluronidase: Role of Peptid vs. Carbohydrate Epitopes and Specific Cross-Reactivity with Hyaluronidase from Honeybee Venom. J Allergy Clin Immunol 2008;121:S30

RATIONALE: Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in YJ allergy and shows 50% sequence homology with hyaluronidase from honeybee venom (Api m 2). As IgE-binding to YJHYA and cross-reactivity with Api m 2 has been recently shown to be due to cross-reactive carbohydrate determinants, the significance of YJ-HYAas a true allergen needs to be re-evaluated. METHODS: Sera from 105 patients with an allergic reaction after a YJ sting (33 YJ CAP single-positive, 72 YJ-honeybee CAP double-positive) were selected and IgE-binding to electrophoretically separated Vespula venom allergens (mixture of V. vulgaris and germanica) was studied by Western blot. To discriminate between IgE-binding to YJ-HYA carbohydrate and peptide epitopes, Western blot inhibition was performed using MUXF-BSA (a synthetic neoglycoprotein consisting of isolated bromelain glycans attached to BSA) and purified Api m 2 as inhibitors. RESULTS: Among double-positive sera, 87% (63/72) reacted with YJHYA. IgE-binding was entirely due to YJ-HYA carbohydrate in 70% (39/ 56), 21% (12/56) showed a mixed antibody response to both peptide and carbohydrate epitopes, and 9% (5/56) reacted with the HYA peptide backbone alone. Nearly half of the sera reacting with YJ-HYA peptide epitopes (8/17) cross-reacted with Apim2. Among YJ single-positive sera, only 9% (3/33) bound with YJ-HYA. CONCLUSIONS: YJ-HYA is only a minor allergen in YJ allergy and sensitization is primarily found in doube-positive patients. Cross-reactivity with honeybee venom HYA (Api m 2) can be observed in approximately 50% of cases.

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[69] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[70] - Rueff F, Werfel S, Przybilla B. Change of the serum concentration of Hymenoptera venom-specific IgE antibodies after a systemic sting reaction - a possible diagnostic tool ? EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°312

Up to 50 % of patients with a history of an anaphylactic sting reaction exhibit specific serum IgE antibodies (sIgE) to both honey bee venom (HBV) and yellow jacket venom (YJV). In some patients it is not possible to distinguish between HBV or YJV allergy by any diagnostic method, and thus treatment with both venoms is indicated. We investigated the change of the serum concentration of venom-sIgE during the first weeks after an anaphylactic sting reaction in order to examine whether this could constitute an additional diagnostic parameter. Inclusion criteria were: 1) diagnosis of YJV (n=31) allergy by routine diagnostic methods; 2) first examination (CAP-FEIA) for sIgE to YJV and HBV 1 - 28 days after an anaphylactic sting reaction, then at least one further examination 2 to 24 weeks after the sting. 31 patients (14 males, 17 females, 9 - 77 years old) were included. An increase or decrease of > 10% of the first measured sIgE level was defined as change. sIgE to YJV was found in all, to HBV in 17 / 31 patients at least at one examination. In 22 patients (71.0 %) an increase, in 7 patients (22.7 %) a decrease, and in 2 patients no change of the serum concentration of sIgE to YJV after the sting reaction was found. The concentration of sIgE to HBV increased in 11 (45.5 %) and decreased in 4 (12.9 %) patients. When the 2nd blood sample was taken within £4 weeks / >4 weeks after the sting, an increase of sIgE to YJV was found in 16/19 (84.2 %) / 6/12 (50.0%; p=0.05). With regard to sIgE to HBV, the respective figures were 8/19 (42.1%) / 3/12 (25.0%; n.s.). Shortly after a sting reaction an increase of sIgE to the relevant insect can be found in a high percentage of patients. In part, also changes of the concentration of possibly cross-reacting IgE-antibodies to the non-causative venom will occur. Cautious interpretation of these changes of sIgE may give additional clues to the offending insect.

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[71] - Straumann F, Bucher C, Wüthrich B. Double sensitization to honeybee and wasp venom: immunotherapy with one or both venoms ? Int Arch Allergy Immunol 2000;123:268-274

BACKGROUND: Double sensitization to honeybee (Apis mellifera) and wasp venom (Vespula spp.) as determined by skin test and measurement of specific IgE is common in hymenoptera sting allergy. Double-sensitized patients have either distinct antibodies for each venom or cross-reacting antibodies that recognize similar or identical epitopes in both venoms. Unfortunately, patients often fail to identify the stinging insect which makes it difficult to distinguish cross-reactors from non cross-reactors. However, for economic reasons as well as for the benefit of the patients, it would be useful to identify complete cross-reactors . METHODS: In this study we investigated 24 double-sensitized patients who were candidates for venom immunotherapy. Homologous and heterologous FEIA inhibition was carried out with honeybee (Apis mellifera) and wasp venom (Vespula spp.) preparations from two different providers. The inhibitor concentrations were ranging from 0 to 100 microg protein/ml . RESULTS: Sera of 4 patients were completely cross-reacting for one venom (3 honeybee, 1 wasp), 8 patients were partially cross-reacting and 10 patients were not cross-reacting. Two patients were excluded from the study due to insufficient homologous inhibition. Data from specific IgE measurements, skin test, and clinical history were not useful for the identification of cross-reacting patients . CONCLUSION: FEIA inhibition is easy to perform and useful for the identification of patients with complete cross-reactivity. In these patients immunotherapy might be restricted to one venom which is beneficial for the patient and cost-effective.

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[72] - Caruso B, Bonadonna P, Severino MG, Manfredi M, Dama A, Schiappoli M, et al. Evaluation of the IgE cross-reactions among vespid venoms. Allergy 2007;62:561-564

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[73] - Paleologou N, Kallergi K, Antonaki G, Marmarinos A, Gouriotis D, Foustoukou M. Cross reactivity between honeybee and yellow jacket in children with hymenoptera allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°402

Background: Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD). Cross-reactivity with specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, may induce positive in vitro results without clinical significance. In the present study, we investigated cross-reactivity between honeybee and yellow jacket in children with hymenoptera allergy. Methods: Skin prick tests (SPT) and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were carried out in 28 stinging-insect allergic patients, aged 6 months to 14 years. All patients reported systemic anaphylactic reactions. After testing, patients were divided in three groups : (a) single positive HBV (n=8), (b) single positive YJV (n=8) and (c) double positive serum (n=12). (CAP class of 3 or higher). RAST inhibition experiments were performed with whole body extracts of HBV and YJV used for venom immunotherapy (Pharmalgen, Denmark). Results: HBV and YJV extracts were able to completely inhibit IgE binding to one of the venoms in two patients with double sensitivity. The prevalence of cross-reactive CD (CCD) was 16.67%. CCD positive patients had a higher total IgE than CCD negative patients. Conclusions: Our data demonstrate that carbohydrate-specific IgE is a frequent cause for in vitro double positivity to HBV and YJV in patients with hymenoptera allergy.

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[74] - Bonifazi F, Jutel M, , Bilò MB, Birnbaum J, Muller U, Bucher C, et al. Prevention and treatment of hymenoptera venom allergy. Allergy 2005;60:1459-1470

Based on the knowledge of the living conditions and habitat of social Aculeatae a series of recommendations have been formulated which can potentially greatly minimize the risk of field re-sting. After a systemic sting reaction, patients should be referred to an allergy specialist for evaluation of their allergy, and if necessary venom immunotherapy (VIT). An emergency medical kit should be supplied, its use clearly demonstrated and repeatedly practised until perfected. This should be done under the supervision of a doctor or a trained nurse. Epinephrine by intramuscular injection is regarded as the treatment of choice for acute anaphylaxis. H1-antihistamines alone or in combination with corticosteroids may be effective in mild to moderate reactions confined to the skin and may support the value of treatment with epinephrine in full-blown anaphylaxis. Up to 75% of the patients with a history of systemic anaphylactic sting reaction develop systemic symptoms once again when re-stung. Venom immunotherapy is a highly effective treatment for individuals with a history of systemic reaction and who have specific IgE to venom allergens. The efficacy of VIT in yellow jacket venom allergic patients has been demonstrated also by assessing health-related quality of life. If both skin tests and serum venom specific IgE turn negative, VIT may be stopped after 3 years. After VIT lasting 3-5 years, most patients with mild to moderate anaphylactic symptoms remain protected following discontinuation of VIT even with positive skin tests. Longer term or lifelong treatment should be considered in high-risk patients. Because of the small but relevant risk of re-sting reactions, in these patients, emergency kits, including epinephrine auto-injectors, should be discussed with every patient when stopping VIT.

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[75] - Hemmer W, Focke M, Kolarich D, Dalik I, Götz M, Jarisch R. Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N-glycans as cross-reactive allergens in honeybee and yellow jacket venom. Clin Exp Allergy 2004;34:460-469

BACKGROUND: IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging-insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy . OBJECTIVE: To confirm the role of carbohydrate epitopes in double positivity and to locate the responsible glycoallergens in HB and yellow jacket (YJ) venom by western blot . METHODS: Immunoblot inhibition using HB venom, YJ venom and two glycoprotein sources displaying 1-3-fucosylated N-glycans (i.e. oilseed rape (OSR) pollen, and the synthetic neo-glycoprotein fucosylated/xylosylated N-glycans from bromelain coupled to bovine serum albumin (MUXF-BSA)) as inhibitors were performed with sera from 15 double-positive patients with stinging-insect allergy. Additionally, reactivity with blotted hymenoptera venoms of a carbohydrate-specific rabbit antiserum against OSR pollen was investigated . RESULTS: Major venom glycoallergens binding with carbohydrate-specific human IgE and rabbit IgG were detected in HB venom at 42 (hyaluronidase (HYA)), 46, 65 and 95 kDa, and in YJ venom at 38 and 43 kDa (HYA). Antibody binding to these allergens was completely lost after periodate treatment. Glycans of HB phospholipase were bound by patients' IgE only after protein denaturation. In 10 of the 15 patients the reactivity was with the second venom because of carbohydrates alone. The high-molecular-weight glycoallergens identified in HB venom probably correspond to similar proteins described earlier, including allergens B and C. The 38-kDa YJ allergen might represent a homologue of V mac 3 . CONCLUSIONS: The data confirm the proposed role of carbohydrate-specific IgE in double positivity to HB and YJ venom and shed new light on some previously described minor hymenoptera allergens of uncertain clinical significance. The consideration of carbohydrate-specific IgE may allow to discriminate between patients with potentially relevant and patients with non-relevant double sensitization.

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[76] - Giroux F, Cano Y, Malandain H. Intérêt diagnostique de l’allergène naturel nCyn d 1 (pollen de chiendent digité, Cynodon dactylon). Rev Fr Allergol 2009;49:330

Objectif.ˆ Le chiendent digité, Cynodon dactylon, est une graminée prospérant surtout en climat chaud. Cette graminée appartient à une sous-famille distante de celle des Pooïdées (dactyle, fléole. . .), de sorte qu‚elle est parfois ajoutée aux Pooïdées dans le protocole de désensibilisation. Il est depuis peu possible de tester in vitro l‚IgE-réactivité vis-à-vis de nCyn d 1, un allergène du chiendent, homologue de Phl p 1 (fléole), etc. Ce test permet-il de préciser le diagnostic et d‚éclairer le choix d‚inclure ou non Cynodon dans le protocole de désensibilisation ? Méthodes.ˆ L‚étude a porté sur 18 patients polliniques aux graminées. La technique CAP Phadia1 a été utilisée pour mesurer l‚IgE-réactivité à nCyn d 1, à rPhl p 1 et à un témoin d‚IgE-réactivité vis-à-vis des épitopes glucidiques (CCD), la broméline. L‚interférence due aux IgE anti-CCD a été évaluée à l‚aide d‚une méthode d‚immuno-absorption et en étudiant également 3 sujets allergiques aux venins d‚hyménoptères et positifs en broméline. Résultats.ˆ Tous les patients polliniques sont positifs pour nCyn d 1,même après immuno-absorption des IgE anti-CCD. L‚allergène Cyn d 1 croise donc avec les allergènes homologues du groupe 1 des graminées, comme Phl p 1. Et si le patient présente une IgE-réactivité vis-à-vis des CCD, la réponse en nCyn d 1 est interférée, surtout quand le rapport rPhl p 1ˆbroméline est faible. Conclusion.ˆ Il semble difficile de tirer parti du test in vitro pour nCyn d 1 en France où les pollens de chiendent digité sont très minoritaires parmi les pollens de graminées : en effet, une IgE-réactivité à nCyn d 1 sera avant tout le reflet de la sensibilisation aux allergènes dominants dans l‚environnement (Phl p 1 fléole, Dac g 1 dactyle, Lol p 1 ivraie. . .) et ne permettra pas de juger s‚il est utile ou non d‚associer Cynodon dactylon dans le protocole de désensibilisation du patient. De plus, le test pour nCyn d 1 est interféré par la présence d‚IgE anti- CCD.

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[77] - Golden DBK. Insect sting allergy and venom immunotherapy: A model and a mystery. J Allergy Clin Immunol 2005;115:439-447

Whole-body extracts of Hymenoptera were used for diagnosis and treatment until controlled clinical trials proved them no better than placebo, whereas venom is 85% to 98% effective. Studies of natural history reveal why whole-body extracts were thought to work. The chance of future systemic reactions is low in large local reactors and in most children and varies between 20% and 70% in adults. Venom skin tests are most accurate, but RAST is an important complementary test. The degree of sensitivity on skin tests or RASTs does not reliably predict the severity of a sting reaction. Venom immunotherapy is recommended for patients at high risk for sting reactions. Rapid regimens are as safe as slower regimens. The recommended dose is 100 microg, but some patients require higher doses for full protection. Venom immunotherapy is continued every 4 to 8 weeks for at least 5 years in most cases. Skin test results become negative in only 25% after 5 years of therapy but in 60% to 70% after 7 to 10 years. When treatment is stopped after 5 years or more, there is a 10% chance of systemic reaction to each future sting, but most reactions are mild. Some patients have a higher risk of relapse and should continue treatment for an extended period.

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[78] - Rieger-Ziegler V, Rieger E, Kranke B, Aberer W. Hymenoptera venom allergy: time course of specific IgE concentrations during the first weeks after a sting. Int Arch Allergy Immunol 1999;120:166-168

Detection of IgE antibodies specific to honeybee or Vespula venoms is an important criterium firstly for the diagnosis of sensitization and secondly for the indication for a specific immunotherapy. Some authors recommend to postpone blood analysis after an insect sting for a certain time because circulating IgE antibodies might be consumed by the allergic reaction, which would result in a false-negative test outcome. We investigated IgE concentrations during the first weeks after an insect sting in 31 patients with an unequivocal history of an anaphylactic reaction after a honeybee (n = 13) or Vespula (n = 18) sting. Blood samples for analysis of specific IgE concentrations (CAP system, Pharmacia Diagnostics, Sweden) were collected within 2 weeks and 5+/-2 weeks after the insect sting. 12/13 patients with honeybee venom and 14/18 patients with Vespula venom sensitization had CAP classes 1 or higher within the first 2 weeks. Those 5 patients with CAP class 0 within the first 2 weeks had detectable IgE concentrations a few weeks later. We conclude that testing for specific IgE to hymenoptera venoms is in most cases useful even during the first 2 weeks after the hymenoptera sting. This allows early decisions on further diagnostic procedures and the therapeutic way to choose. Patients with no detectable IgE should, however, be retested after a few weeks.

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[79] - Bilò BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JNG, et al. Diagnosis of hymenoptera venom allergy. Allergy 2005;60:1339-1349

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

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[80] - Birnbaum J. Allergie aux venins d'hyménoptères. Qui, comment et combien de temps désensibiliser ? Rev Fr Allergol Immunol Clin 2005;45:489-492

Si la sensibilisation aux venins d'hyménoptères est fréquente, l'allergie est plus rare mais se retrouve chez 1 à 3 % de la population générale. Sa gravité clinique, tel le choc anaphylactique, est l'indication immédiate d'une désensibilisation. Les manifestations légères cutanéomuqueuses ne relèvent pas a priori d'une désensibilisation. La connaissance de facteurs de risque à la récidive ou à l'aggravation d'une nouvelle réaction en cas de piqûre permet d'adapter l'indication à chaque patient. Quel que soit le protocole de désensibilisation utilisé, en sachant que les protocoles rush ou ultrarush sont actuellement les plus prescrits et conseillés, des réactions secondaires sont observées. Peu de facteurs sont connus comme permettant d'identifier le patient à risque d'une mauvaise tolérance lors de la désensibilisation. Les antihistaminiques ne préviennent que les réactions locorégionales et les réactions générales minimes. Leur rôle éventuel comme agent pouvant augmenter l'efficacité de la désensibilisation les fait conseiller pendant les phases de progression des doses de venin. En dehors de certaines situations, la dose de rappel doit être au minimum de 100 µg mais parfois elle devra être de 200 µg. La durée de la désensibilisation sera au minimum de cinq ans. Elle pourra être arrêtée au bout de trois ans si le bilan biologique (tests cutanés et IgEs) est négatif et sera poursuivi au-delà de cinq ans dans certaines situations à risque.

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[81] - Bilò MB, Brianzoni MF, Cinti B, Napoli G, Fusari A, Bonifazi F. The dilemma of the negative skin test reactors with a history of venom anaphylaxis. Allergy Clin Immunol Int 2005;17(Suppl. 1):69

The loss of sensitization over time, the involvement of a different pathogenetic mechanism and the poor sensitivity of diagnostic tests have been included among the causes of a non IgE-mediated anaphylaxis triggered by an insect sting. To provide further insight into this topic we describe the case of a patient monosensitive to Polistes dominulus venom. The patient is a 38-year-old man suffering from urticaria pigmentosa, with a history of anaphylactic shock to a Vespid sting in 1997 (identified as Polistes). In another Allergy Unit in 2000, CAP-System (Pharmacia S.p.A) for Vespula germanica, Polistes sp., Vespa crabro and Apis mellifera venoms was negative. As a result, the patient was diagnosed with "non-IgE mediated insect sting anaphylaxis in subjects with urticaria pigmentosa". A self-administration emergency kit was prescribed and standard patient guidelines provided on how to avoid a further sting. The patient came to our notice in 2003 and prick and intradermal tests with Vespula sp., Polistes sp., Apis mellifera venoms were negative. The tryptase serum level was 27 mcg/L (v.n. < 11,5 mcg/L). Since cross-reactivity between the European species of Polistes is very high, but weaker between the American and European species, serum specific IgE (CAP-System Pharmacia S.p.A.) to Polistes dominulus venom was tested for and yielded negative results. The intradermal test with Polistes dominulus venom (Anallergo ˆ Florence) was positive (0.1 mcg/ml). "Ultra-rush" specific immunotherapy using an acqueous Polistes dominulus venom extract (Anallergo - Florence) was performed over two mornings until a total dose of 100mcg was reached without any side effects. Presently the patient is undergoing maintenance therapy (depot Polistes dominulus venom extract, Anallergo - Florence) withouth side effects and has not been re-stung. In conclusion, despite the high cross-reactivity among the venoms of the various species of Polistes, recent evidence of a selective specificity unique to European Polistes allergens raises the problem of the need to make the American Polistes venom as well as that of Polistes dominulus (a very common insect in Europe) available both for diagnostic and therapeutic purposes. Therefore this case history demonstrates that omitting to perform skin tests for Polistes dominulus venom would have led to the same misdiagnosis of non IgE-mediated anaphylaxis and served to further delay commencing specific immunotherapy.

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[82] - Bilo MB, Brianzoni F, Cinti B, Napoli G, Bonifazi F. The dilemma of the negative skin test reactors with a history of venom anaphylaxis: will this always be the case ? Eur Ann Allergy Clin Immunol 2005;37:341-342

The loss of sensitization over time, the involvement of a different pathogenetic mechanism and the poor sensitivity of diagnostic tests have been included among the causes of a non IgE-mediated anaphylaxis triggered by an insect sting. To provide further insight into this topic we describe the case of a patient suffering from urticaria pigmentosa, anaphylactic shock due to a Vespid sting and monosensitive to Polistes dominulus venom, who was previous diagnosed with non-IgE mediated insect sting anaphylaxis in subjects with urticaria pigmentosa. Therefore ultra-rush specific immunotherapy using an acqueous Polistes dominulus venom extract was performed over two mornings until a total dose of 100mcg was reached without any side effects. Presently the patient is undergoing maintenance therapy with a depot Polistes dominulus venom extract at 4-weekly intervals and is free of side effects. In conclusion, despite the high cross-reactivity among the venoms of the various species of Polistes, recent evidence of a selective specificity unique to European Polistes allergens raises the problem of the need to make the American Polistes venom as well as that of Polistes dominulus (a very common insect in Europe) available both for diagnostic and therapeutic purposes. Besides the fact that the presence of urticaria pigmentosa alone could have explained the anaphylactic reaction to the aspecific stimulation triggered by the venom, this case history also demonstrates that omitting to perform skin tests for Polistes dominulus venom would have led to the same misdiagnosis of non IgE-mediated anaphylaxis and served only to further delay commencing specific immunotherapy.

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[83] - Cretin JY. Hyménoptères d'Europe et du bassin méditerranéen. Rev Fr Allergol Immunol Clin 2006;46:274-276

Parmi les hyménoptères, seuls les aculéates (latin aculeatus « muni d'un aiguillon ») sont susceptibles d'infliger des piqûres grâce à leur dard venimeux. Tout en restant dans le domaine européen, la diversité spécifique de ce groupe est passée rapidement en revue, avec un rappel des familles classiquement connues pour provoquer des réactions allergiques (insectes sociaux : apidés, vespidés, formicidés), et une évocation des familles non sociales qui, fortes de plusieurs milliers d'espèces bien plus discrètes, entrent parfois en contact accidentellement avec l'homme. Il est en effet hautement probable que certaines réactions atypiques ou non reliées ensuite à des venins connus soient dues à ces hyménoptères solitaires, rencontrées fortuitement au cours des diverses activités humaines, à la campagne comme dans les secteurs fortement urbanisés.

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[84] - Rueff F, Bauer C, Albert K, Przybilla B. Hymenoptera sting anaphylaxis without venom sensitization is no indication of mastocytosis. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°72

Background: Sporadically, patients with mastocytosis and a history of an anaphylactic sting reaction, but without sensitization to honeybee or yellow jacket venoms (Hymenoptera venoms, HV) were reported. It was speculated that HV could induce mast cell release by its toxic action and thus mastocytosis - with an increased mast cell number - is a risk factor for pseudo-allergic sting anaphylaxis. An elevated baseline serum concentration of mast cell tryptase (BSCT) is frequently associated with mastocytosis. We assessed the BSCT in patients who had no indication of HV sensitization despite a history of insect sting anaphylaxis. Methods: Patients who presented between 1999 and 2003 because of a history of Hymenoptera sting anaphylaxis and who had no demonstrable HV sensitization were included into the evaluation. The absence of HV sensitization was stated if no skin prick test reaction to 100 µg HV / ml, no intradermal test reaction to 1 µg HV / ml, and no HV-specific serum IgE antibodies (CAP-FEIA) were demonstrable. Severity of sting reactions was classified as grade I if there was a history of generalised skin symptoms, as grade II in the case of minor to moderate respiratory and/or cardiovascular symptoms, or as grade III if full shock had developed. BSCT was measured in stored frozen sera (UniCAP Tryptase). According to the manufacturer, the 95th percentile of BSCT in the serum is 11.4 µg/l, the geometric mean being 3.8 µg/l. Further examinations for mastocytosis were not performed. Results: 19 patients (3 males, 16 females; 34.3 ± 18.2 years old) fulfilled the inclusion criteria. Previous sting reactions were grade I in 2, grade II in 14, and grade III in 3 patients. The mean interval between sting reaction and diagnostics was 54.6 ± 76 (1-221) months. BSCT was 1.0 - 9.4 µg/l, the geometric mean was 4.4 µg/l. Conclusion: An elevated BSCT was not found in any of the patients without demonstrable HV sensitization despite Hymenoptera sting anaphylaxis. This finding in a rather large group of patients rules out a strong relationship between "test-negative" Hymenoptera sting anaphylaxis and mastocytosis, even if BSCT is not always elevated in patients with mastocytosis.

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[85] - Bilò BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JNG, et al. Diagnosis of hymenoptera venom allergy. Allergy 2005;60:1339-1349

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

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[86] - Hoffman DR. Hymenoptera venom allergens. Clin Rev Allergy Immunol 2006;30:109-128

Hymenoptera venoms each contain a variety of protein allergens. The major components have all been characterized, and most of the amino acid sequences are known. This article concentrates on the use of contemporary techniques including cloning, mass spectrometry and genomics in the characterization of venom allergens, and newer separation techniques for protein isolation. Examples of the use of these techniques with venom proteins are presented.

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[87] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[88] - Rendic D, Klaudiny J, Stemmer U, Schmidt J, Paschinger K, Wilson IB. [Towards abolition of immunogenic structures in insect cells]. Biochem J 2007;402:105-115

Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of alpha1,3-fucosylation: the modification by alpha1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcbeta1,4(Fucalpha1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active alpha1,3-FucTs (alpha1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core alpha1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Le(x) [Galbeta1-4(Fucalpha1-3)GlcNAc] epitope in vitro and is the first Lewis-type alpha1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed alpha1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance

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[89] - Kolarich D, Léonard R, Hemmer W, Altmann F. The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris. FEBS J 2005;272:5182-5190

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.

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[90] - Seppälä UR, Mutenda K, Monsalve R, Skov L, King TP, Ipsen H, et al. Analysis of N-Glycosylation in Vespula Vulgaris Hyaluronidase Ves v 2. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°465

RATIONALE: Glycosylated venom allergens have been identified to cause in vitro double positivity in patients allergic to yellow jackets and bees. Vespula vulgaris hyaluronidase, Ves v 2, is one of the major venom glycoallergens, which has been shown to carry carbohydrate determinants binding cross-reactive IgE. The glycosylation patterns, however, remain unresolved METHODS: Natural Ves v 2 was subjected to reversed-phase chromatography following enzymatic digestions by trypsin, Asp-N and/or chymotrypsin. Mass spectrometry (MS) was used to study peptide sequences and to identify putative glycopeptides. Enzymatic degradation followed by MS was used to demonstrate N-glycosylation sites in Ves v 2. The N-glycosylation sites were then modelled onto the structure of Ves v 2 RESULTS: MS analysis of Ves v 2 revealed a broad peak in the m/z range from 41455.64 to 43703.10. Further analyses of Ves v 2 peptides by MS revealed five peptides diagnostic of glycosylation. Enzymatic cleavage of these glycopeptides by N-glycosidase revealed three peptides corresponding to N- glycosylation at Asn79 and 127. Enzymatic cleavage by Nglycosidase A revealed two peptides corresponding to N-glycosylation at Asn 99. Interestingly, MS analyses also demonstrated that Asn79 and 99 can also be expressed without N-glycan(s).

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[91] - Winningham KM, Fitch CD, Schmidt M, Hoffman DR. Hymenoptera venom protease allergens. J Allergy Clin Immunol 2004;114:928-933

Background Recent studies have shown the presence of additional allergenic proteins in honeybee and paper wasp venoms. Both venoms contain serine protease enzymes. Objective We isolated and obtained complete sequences of honeybee and Mediterranean paper wasp venom proteases, both of which have significant IgE binding activity. The structures are compared with bumblebee venom protease. Methods Venom proteases were chromatographically isolated from venoms and partial amino acid sequences determined. RT-PCR and rapid amplification of cDNA ends methods were used to clone cDNA, and complete sequences were determined for honeybee and a paper wasp venom protease. Results The venom proteases are all serine proteases of the trypsin type. The honeybee protease contains a complement, embryonic sea urchin protein, bone morphogenetic protein interaction domain as well as a linker and propeptide sequence, and a unique methionine residue near the active site. It has IgE binding activity. The paper wasp protease is a single trypsin domain and is an important allergen. The framework residues are poorly conserved among honeybee, bumblebee, and paper wasp enzymes. Conclusions The 3 venom serine proteases have significant IgE binding activities. The structures are poorly conserved even among the Apidae , suggesting little cross-reactivity among the protein portions. The paper wasp venom proteases are important allergens.

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[92] - Fitch CD, Schmidt M, Hoffman DR. Carbohydrates are Important in IgE Binding to the Venom Serine Protease from Polistes dominulus. J Allergy Clin Immunol 2008;121:S29

RATIONALE: A serine protease has been identified as a significant allergen in the venom of Polistes dominulus. Previous studies with this protein suggest that carbohydrate may play a role in its ability to bind IgE. METHODS: The previously cloned serine protease sequence was used to produce recombinant protease in both prokaryotic and eukaryotic systems. A construct was designed for the baculovirus system which contained the propeptide and mature peptide sequence (BVP). Two constructs were used in an E. coli system; one contained the sequence for the propeptide and mature molecule (P3), while the second contained the sequence for the mature molecule only (P2). These recombinant proteins were purified using affinity chromatography and their identity confirmed with MALDI-TOF mass spectrometry. Their immunological reactivity were analyzed using regular and competitive immunoblot experiments. RESULTS: Immunoblotting using sera from sensitized individuals, showed 100% positive for IgE-binding to BVP and 20% of sera displayed weak binding to P2. Immunoblots competitively inhibited with bromelain showed decreased binding to BVP. CONCLUSIONS: Immunological tests with BVP, P2 and P3 demonstrated IgE binding to the glycoslyated protease, while the unglycoslyated form reacted weakly to some sera. Competitive assays with carbohydrate, showed a marked decrease in binding activity to the glycoslyated form, but not complete inhibition. This data suggests that carbohydrate is an important epitope for this allergen, however, further investigation is needed into the role of other conformational and sequential epitopes in IgE binding to this protease.

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[93] - Barboni E, Kemeny DM, Campos S, Vernon CA. The purification of acid phosphatase from honey bee venom (Apis mellifica). Toxicon 1987;25:1097-1103

Acid phosphatase from bee venom was purified by a combination of saturated ammonium sulphate precipitation, gel filtration and ion exchange chromatography. The final product which is a glycoprotein contained less than 0.1% phospholipase A2 or hyaluronidase activity and existed in two molecular weight (96,000 and 45,000) forms. Acid phosphatase is a potent allergen, in bee venom allergic patients, which is capable of releasing histamine from sensitized human basophils and of inducing a wheal and flare reactions in sensitized human skin

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[94] - Blank S, Seismann H, Braren I, Greunke K, Cifuentes L, Grunwald T et al. Dissecting CCD reactivity in hymenoptera venom allergy by diminution of alpha-1,3-core fucosylation. Allergy 2009;64(Suppl. 90):39

Background: To date, diagnosis of hymenoptera venom allergy using in vitro tests is employed routinely, but remains severely hampered by cross-reactivities and false-positive test results. In general, about 30-40% of patients with insect venom allergy have IgE antibodies reacting with both honeybee and wasp venom. Apart from true double sensitization, IgE against crossreactive carbohydrate determinants (CCD) are the most frequent and often only cause for multiple reactivity found in insect venom allergy. Methods: In order to address the challenge of allergenic cross-reactivity and double sensitization we used recombinant approaches employing cell lines with variant capacities of a-1,3-core fucosylation, the causative crossreactive glycostructure on hymenoptera venom allergens. Results: The venom hyaluronidases and supposed major allergens Api m 2 and Ves v 2 as well as a human control protein were recombinantly produced in native form in different types of insect cells. In contrast to T. ni cells absence or immunologically negligible presence of a-1,3-core fucosylation upon production in S. frugiperda cells could be proven for both allergens and the reference protein employing a CCD-reactive rabbit serum as well as sera of individuals with anti-CCD IgE. The specificity of this effect further could be verified by de novo establishment of CCD reactivity upon coexpression of honeybee a-1,3-fucosyltransferase in S. frugiperda cells. Conclusion: Employing the set of proteins with or without CCDs evaluation of serum reactivities of venom-sensitized individuals could be performed. The use of such recombinant molecules represents a novel strategy with implications for diagnostic and therapeutic approaches.

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[95] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[96] - Mairesse M, Ledent C. Allergie aux venins d'hyménoptères: intérêt du dosage des IgE anti-broméline. Rev Fr Allergol Immunol Clin 2003;43:95

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[97] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[98] - Jappe U, Hoffmann M, Burow G. Potential screening allergens for detection of carbohydrates as cause for in vitro double positivity to honeybee and yellow jacket venom as well as rubber latex in patients with stinging insect allergy. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°69

IgE-double positivity for honeybee (HB) and yellow jacket (YJ) venom sometimes causes diagnostic difficulties concerning therapeutical strategies. True double sensitisation may account for some of the results. Another explanation is crossreactivity caused by sequence homologies between the venom hyaluronidases. However, previous investigations revealed that polyreactive human sera binding through carbohydrates with HB venom also bind to a broad range of pollen and food allergens, and, according to few reports, to rubber latex as well. In this study, 98 patients, who attended the clinic between 1996-2003 with stinging insect allergy and additionally were CAP RAST positive for both, HB and YJ IgE, were investigated for carbohydrate-binding IgE. The following allergens were used in the CAP-RAST: HB, YJ, whole extracts of timothy pollen, rape pollen, and rubber latex, bromelain, and horse radish peroxidase, all carrying glycans, except two additional allergens, the recombinant timothy pollen components Phl p1 and Phl p5, which were produced by expression in E. coli without a glycosylation step. 26/98 patients with CAP RAST-positivity to both venoms did not show IgE-positivity to other allergens, 72/98 additionally reacted to carbohydrate-carrying allergens. 52 out of these did not detect the recombinant components Phl p1 and Phl p5, and 20/72 bound with the whole range of allergens. Out of 72 carbohydrate-reactive sera, 20 were binding to rubber latex. For 20 sera, an additional reciprocal inhibition assay was performed with HB and YJ venom, respectively. 16/20 were negative for carbohydrate-binding IgE, 6/16 showed a homologous but not heterologous reaction, indicating a true double sensitisation of those patients. In cases of IgE double positivity to both insect venoms supplementary CAP RAST with at least one carbohydrate containing allergen plus recombinant allergens, e.g. timothy pollen allergen plus its recombinant components and/or horse radish peroxidase should be performed. The detection of carbohydrate-binding IgE as well as a subsequent reciprocal inhibition assay are essential diagnostic tools to specify initial CAP RAST results.

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[99] - Jappe U, Hoffmann M, Burow G, Enk A. Pitfalls of in vitro-allergy diagnostic: glycan-associated epitope sharing of insect venom and natural rubber latex allergens. Significance of screening allergens, reciprocal inhibition, and recombinant allergens. Allergy Clin Immunol Int 2005;17(Suppl. 1):191-192

Background: IgE-double positivity for honeybee (HB) and yellow jacket (YJ) venom may be due to true double sensitization, artefacts caused by increased sensitivity of the test system and cross-reactive carbohydrate determinants (CCD). It may cause diagnostic difficulties concerning therapeutical strategies. Previous investigations revealed that IgE-positive human sera for both, HB and YJ venom, also bind to a broad range of plant allergens due to CCD. Sera of patients with insect venom allergy who had attended the department between 1995 and 2004 were evaluated. Methods: 146 patients with suspected stinging insect allergy and CAP FEIA-double positivity were investigated for specific (s)IgE to additional CCDcontaining allergens: whole extracts of timothy pollen, rape pollen, rubber latex, bromelain, and horse radish peroxidase (HRP). Sera positive for rubber latex IgE were further investigated with the recombinant latex components rHev b1, b2, b3, b5, b6.01, b6.02, b8, b9, and b11. The corresponding patients were further investigated for clinical relevance of the CAP-FEIA-results (questionnaire, skin prick test). Reciprocal inhibition assays with both venoms (100µg/ml) and HRP (500µg/ml) were performed. Results: 38/146 patients were sIgE-positive to both venoms only. 108/146 additionally reacted to CCD-carrying allergens. 91/108 CCD-reactive sera had sIgE to rubber latex. 37/56 rubber latex-IgE-positive sera were negative for anti-recombinant-latex-IgE, 19/56 were positive for at least one recombinant component. 21/56 of the corresponding patients who were already available for a skin prick test with natural rubber latex and a questionnaire were negative in history and skin prick test. An additional reciprocal inhibition assay was performed for 29 sera with HB and YJ venom, respectively. 8/29 showed a homologous but not heterologous reaction, indicating a true double sensitization of those patients. Inhibition with HRP in 24 sera revealed 100% inhibition of anti-HRP-IgE binding, in 3/24 HRP completely inhibited IgE-binding to YJ, and in 11/24 sera to HB. Conclusions: In cases of IgE-positivity to both insect venoms supplementary CAP FEIA with at least one CCD containing allergen as screening test should be performed. Subsequent reciprocal inhibition is an essential diagnostic tool to specify cross-reacting CAP FEIA results as will most probably be recombinant allergens after having been evaluated for their specificity and clinical relevance.

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[100] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[101] - Jappe U, Hoffmann M, Huebsch-Mueller C, Enk A, Raulf-Heimsoth M. Specific IgE-antibody reaction to cross-reactive carbohydrate determinants (CCD) in hymenoptera venoms: comparison of two different in vitro-tests. Allergy 2007;62(suppl. 83):50

Background: IgE-positivity for both hymenoptera venoms is mostly due to cross-reactive carbohydrate determinants (CCD). This study evaluated CCD-IgE-binding intensity in two in vitro-IgE-detection-methods. Patients and methods: 56 patients with stinging insect allergy and anti-CCD-sIgE were investigated with further CCD-allergens: timothy grass (Phl p), natural rubber latex (NRL), bromelain (BRO), and horse radish peroxidase (HRP) in 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (DPC Biermann, Germany), based on liquid phase technology. The new CCD-ImmunoCAP, a MUXF3-type carbohydrate and ascorbate oxidase (DPC), also a glykan-containing allergen, were used as well. The sera were further investigated via hymenoptera venom- immunoblot (DPC). Results: All sera had sIgE to both insect venoms. In CAP FEIA, 47/56 (83.9%) had sIgE to NRL, 52/56 (92.9%) to Phl p, 50/56 (89.2%) to BRO, 50/56 (89.2%) to HRP and 48/56 (85.7%) to CCD-CAP. In Immulite, 30/56 (53.6%) had sIgE to NRL, 40/56 (71.4%) to BRO, 46/56 (82.1%) to HRP, 40/56 (71.4%) to Phl p, and 45/56 (80.4%) to ascorbate oxidase. In CAP FEIA sIgE-concentrations to BRO and HRP, showed a strong Pearson correlation with 0.9994 (p<0.0001). Although they differed individually in Immulite, the Pearson correlation was still good (0.6953; p=0.01). However, via Immulite, HRP-sIgE could be measured in 8 subjects, where no BRO-sIgE were detected, whereas only 2 individuals had sIgE to BRO and none to HRP. Regarding the class-grading, the differences between BRO and HRP in Immulite measured were 3 classes in 3, 2 classes in 20, and 1 class in 16 subjects. This phenomenon is dependent on BRO-DPC and is mirrored by a non-significant Pearson correlation for BRO-DPC/BRO-CAP (0.488; p=0.56), BRO-DPC/HRP-CAP (0.497; p=0.51) and a significant Pearson correlation for HRP-DPC/BRO-CAP (0.8905; p<0.0001). In immunoblot, all sera reacted with the glycosylated allergens. As in CAP FEIA the IgE-binding to the CCD structures was detected in more sera and was comparable with all the tested CCD allergens, it might be assumed that these epitopes are equally presented on the different ImmunoCAPs. The differences in the IgE-binding to BRO and HRP in the Immulite E 2000 system might be due to their presentation in these allergens in liquid phase. However, HRP seems to be the favourable screening allergen in cases of IgE-double-positivity in insect venom allergic patients.

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[102] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[103] - Hemmer W, Jin C, Focke M, Jarisch R, Altmann F. IgE reactivity with peptid versus carbohydrate epitopes on yellow jacket venom hyaluronidase and specific cross-reactivity with hyaluronidase from honeybee venom. Allergy 2007;62(suppl. 83):112

Background: Yellow jacket venom hyaluronidase (YJ-HYA) is considered a major allergen for patients with YJ allergy and shows 50% sequence homology with honeybee venom hyaluronidase (Api m 2). As IgE-binding to YJ-HYA and cross-reactivity with Api m 2 has recently been shown to be due to cross-reactive carbohydrate determinants, the significance of YJ-HYA as a clinically relevant allergen needs to be re-evaluated. Method: Sera from patients with allergic reactions after YJ stings and a double-positive CAP result for both Vespula and honeybee venom were selected and IgE-binding to electrophoretically separated Vespula venom allergens (mixture of V. vulgaris and germanica) was studied by Western blot. To discriminate between IgE-binding to carbohydrate and peptide epitopes, Western blot inhibition was performed in 40 sera positive for YJ-HYA, using MUXF-BSA (a synthetic neoglycoprotein consisting of pure bromelain glycans attached to BSA) and - in some patients - purified Api m 2 as inhibitors. Results: Of 64 sera tested, 49 (83%) bound with YJ-HYA in the Western blot, 55 (85%) with phospholipase, and 49 (75%) with antigen 5. Among patients reacting with YJ-HYA, the IgE-binding was entirely due to carbohydrate in 65% (26/40), 25% (10/40) showed a dual antibody response to both peptide and carbohydrate epitopes, and in 10% (4/40) the IgE was directed against the HYA peptide backbone alone. Serum preincubation with purified Api m 2 in four patients confirmed that specific cross-reactivity between YJ and honeybee HYA does occur also on protein level. Conclusion: In this set of preselected YJ-allergic patients with double-positive CAP results for YJ and honeybee venom, only 30% had IgE reacting with peptide epitopes on YJ-HYA, identifying YJ-HYA as a minor allergen only. The role of YJ-HYA in an unselected population of patients with YJ allergy, and the frequency of cross-reactivity between YJ and honeybee venom HYA remains to be elucidated.

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[104] - Hemmer W, Jin C, Focke M, Jarisch R, Altmann F. Yellow Jacket Venom Hyaluronidase: Role of Peptid vs. Carbohydrate Epitopes and Specific Cross-Reactivity with Hyaluronidase from Honeybee Venom. J Allergy Clin Immunol 2008;121:S30

RATIONALE: Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in YJ allergy and shows 50% sequence homology with hyaluronidase from honeybee venom (Api m 2). As IgE-binding to YJHYA and cross-reactivity with Api m 2 has been recently shown to be due to cross-reactive carbohydrate determinants, the significance of YJ-HYAas a true allergen needs to be re-evaluated. METHODS: Sera from 105 patients with an allergic reaction after a YJ sting (33 YJ CAP single-positive, 72 YJ-honeybee CAP double-positive) were selected and IgE-binding to electrophoretically separated Vespula venom allergens (mixture of V. vulgaris and germanica) was studied by Western blot. To discriminate between IgE-binding to YJ-HYA carbohydrate and peptide epitopes, Western blot inhibition was performed using MUXF-BSA (a synthetic neoglycoprotein consisting of isolated bromelain glycans attached to BSA) and purified Api m 2 as inhibitors. RESULTS: Among double-positive sera, 87% (63/72) reacted with YJHYA. IgE-binding was entirely due to YJ-HYA carbohydrate in 70% (39/ 56), 21% (12/56) showed a mixed antibody response to both peptide and carbohydrate epitopes, and 9% (5/56) reacted with the HYA peptide backbone alone. Nearly half of the sera reacting with YJ-HYA peptide epitopes (8/17) cross-reacted with Apim2. Among YJ single-positive sera, only 9% (3/33) bound with YJ-HYA. CONCLUSIONS: YJ-HYA is only a minor allergen in YJ allergy and sensitization is primarily found in doube-positive patients. Cross-reactivity with honeybee venom HYA (Api m 2) can be observed in approximately 50% of cases.

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[105] - Jappe U, Sander I, Hoffmann M, Huebsch-Mueller C, Enk A, Raulf-Heimsoth M. Cross-reactive carbohydrate determinants and Hymenoptera venom allergy: IgEdetection in conceptually different automated systems, implicating relevant recombinant allergens. Allergy 2008;63(suppl. 88):99-100

Background: Cross-reactive carbohydrate determinants (CCD) are the most widely distributed pan-epitopes seemingly affecting the specificity of IgE-detection methods. Methods: Fifty-six patients with insect venom allergy were investigated in two conceptually different systems with bromelain (BRO), horse radish peroxidase (HRP) and natural rubber latex (NRL): 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (IML, DPC Biermann, Germany), based on liquid phase technology, including the CCDs MUXF3 (Phadia) and ascorbate oxidase (DPC), respectively. Most sera were additionally investigated with recombinant venom allergens in ADVIA CENTAUR. Results 40.35 kU/L were considered positive. The controls were 10 grass pollen allergics. Results and discussion: In contrast to CAP, where all patients‚ sera had IgE to both insect venoms (yellow jacket, YJ; honey bee, HB), in IML only 39/56 had IgE to HB and 52/56 to YJ. Five out of fifty-six had no IgE to any CCD-allergen presented in both systems. Using CAP, 40/51 had BRO-IgE, 45/51 IgE to HRP, 37/51 to NRL, 38/51 to MUXF3, whereas in IML 24/51 had IgE to BRO, 43/51 to HRP, 19/51 to NRL, and 37/ 51 to ascorbate oxidase. In CAP, IgE to BRO and HRP showed a strong Pearson correlation with r=0.9994 (P<0.0001). Although IgE to these CCDs differed individually in IML, the Pearson correlation was still good (0.6953; P<0.01). The sera of grass pollen allergics showed IgE to HB (CAP: 4/10; IML 1/10), to YJ (CAP: 5/10; IML: 6/10), to NRL: 8/10, BRO: 6/10, HRP: 4/10 in CAP; in IML: 9/10 to NRL, 7/10 to HRP, 8/10 to ascorbate oxidase and 6/10 to BRO without clinical relevance, strengthening the hypothesis that grass sensitisation induces anti-CCD-IgE. In ADVIA, 28/33 had IgE to YJ-, 14/31 to HBextract, 11 to both. 12/34 bound to Api m 1, 26/34 to Ves v 5, 8/34 to both. IML-results not corresponding with CAP showed accordance with ADVIA for 6 HB-IgE negatives being clearly positive for YJ in ADVIA, 5/6 being positive for YJ in both systems (IML, ADVIA). The serum only HRP- and BROPoster IgE-positive in IML had YJ-IgE in ADVIA. In 21/34, ADVIA confirmed history, in 6 cases there was no concordance, and where the culprit insect remained unidentified (n=7), ADVIA clearly pointed to one venom, 5 YJ, 2 HB. The differences in the IgE-binding to BRO and HRP in IML in contrast to CAP might be due to the presentation of the CCD-epitopes in liquid phase. HRP is the favourable CCD-screening allergen in both systems.

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

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

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[107] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[108] - Giroux F, Cano Y, Malandain H. Validation of a simple method to overcome the interference of cross-reactive carbohydrate determinants (CCD) in specific IgE assays. Allergy 2009;64(Suppl. 90):8-9

Background: Some patients develop IgE reactivity to glyco-epitopes, ie. carbohydrate chains present on glycoproteins. This is especially the case for patients allergic to pollens or to Hymenoptera venoms. These glyco-epitopes are often cross-reactive, leading to so-called IgE anti-CCD (Cross-reactive Carbohydrate Determinants). Several studies failed to find clinical relevance for these IgE. But in vitro diagnostic tests do not differenciate between peptide- and glycan-specific IgE (sIgE) and their results can be interfered by the presence of IgE anti-CCD in the patient's serum. We sought to design a serum pretreatment to overcome this drawback. We especially aimed at simplifying the process and minimalizing cost and serum dilution so that the method could be suited to most daily routine situations. METHODS: Serum pretreatment: a reagent made of heat-inactivated bromelain and horseradish peroxidase (HRP) was added to the serum. After a waiting time, sIgE reactivity was directly measured on UniCAP (Phadia). To set up the method we used 2 sera positive to IgE anti- CCD (CAP bromelain >0,35 kU/l) (Eur Ann Allergy Clin Immunol 2007;39:216) and a pool of negative sera (total IgE <2 UI/ml). For validating the method, we selected 40 sera from our serum bank, 13 being positive to IgE anti-CCD. We studied 7 allergen sources (peanuts, white of egg, milk, cat ,dog, mite, cod) and 6 non glycosylated recombinant allergens (rBet v 1, rBet v 2, rPhl p 1, rPhl p 7, rPhl p 12, rFel d 1). RESULTS: The best compromise between the highest capture of IgE anti-CCD and the lowest serum dilution was to mix 4 parts of serum with 1 part of reagent (bromelain and HRP 25 g/l, a.a.). Serum pretreatment did not generate false positive IgE-reactivity in negative sera, nor significantly modified positive results for milk, cat, dog, mite, cod, white of egg, and non glycosylated allergens (n = 54 comparisons). It did not modify peanut results for 7 sera with bromelain <0,5 kU/l, and corrected peanut diagnostic for 7 patients with falsely high peanut sIgE (bromelain 1.5-28 kU/l). CONCLUSION: We think our method could be applied to daily lab routine. Components of the pretreatment reagent are ready-to-use, stable (>1 month, 4°C), and cheap (<0.1 •/test). Serum dilution is minimal so that CCD capture can be performed with IgE reactivity as low as 0,5 kU/ l. The method could prove especially useful to CCD-prone sIgE tests such as peanuts, latex and Hymenoptera venoms.

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[109] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[110] - Guerti K, Bridts CH, Stevens WJ, Ebo DG. Wasp venom-specific IgE: towards a new decision threshold ? J Investig Allergol Clin Immunol 2008;18:321-323

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[111] - Mertens M, Brehler R. The clinical relevance of CCDs in hymenoptera-venom allergy. Allergy 2008;63(suppl. 88):24-25

Background: About 20% of allergic patients have specific (s.)-IgE-antibodies against the carbohydrate-part of allergens (cross-reactive carbohydrate determinant5CCD). These glycans are found on a wide variety of allergens like pollens, hymenoptera-venoms and foods. In most cases patients with s.-IgE against CCDs show no clinical symptoms and skin prick tests (SPT) are negative. In sera-tests these antibodies are referred as ŒŒfalse-positive‚‚ results. However, for a small subset of allergens (e. g. a specific tomato-allergen; Lyc e 2) it is shown that CCDs alone have the property to induce histamine-release of basophils passively sensitised with serum of allergic patients. This leads to the assumption that CCDs can have the capability to induce allergic reactions in vivo. Methods: To investigate the role of CCDs in hymenoptera-venom allergy patients‚ serum was analysed for s.-IgE against insect venoms, CCD and horseradish-peroxidase (HRP) - hymenoptera venom allergy was also verified by skin testing and patients‚ history. Furthermore basophils of these patients were stimulated with different concentrations of periodate-treated venomextracts (lacking glycan-epitopes), proteinase K treated wasp venom (lacking proteins epitopes), their native counterparts and HRP. Afterwards activation of basophils was measured by FACS-analysis to get information whether CCDs elicit an activation in vitro or not. Results: Regarding the basophil activation test wasp venom allergic patients with s.-IgE against CCD and HRP can be divided into two subgroups. Subgroup one shows only an activation with wasp venom (native and without glycan-epitopes) and no activation with any honeybee venom, whereas subgroup two shows an additional activation with native honeybee venom (at least in the highest concentration) but not with the glycan-lacking venom. Basophils stimulated with HRP show the same deviation-pattern: basophils of subgroup one show only an activation with high concentrations of HRP, whereas basophils of subgroup two show a stimulation with already about a 100 times less HRP. The same results were found for honeybee venom-allergic patients. Conclusion: We assume that CCDs are relevant in vitro in a subgroup of hymenoptera- venom allergic patients. Currently we analyse the clinical relevance of anti-CCDantibodies in patients with a positive basophil activation test by further skin tests with other glycan-rich allergens and a clinical follow-up.

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[112] - Müller UR, Johansen N, Petersen AB, Fromberg-Nielsen J, Haeberli G. Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m 1 and Ves v 5. Allergy 2009;64:543-548

BACKGROUND: In patients with hymenoptera venom allergy diagnostic tests are often positive with honey bee and Vespula venom causing problems in selection of venoms for immunotherapy . METHODS: 100 patients each with allergic reactions to Vespula or honey bee stings and positive i.e. skin tests to the respective venom, were analysed for serum IgE to bee venom, Vespula venom and crossreacting carbohydrate determinants (CCDs) by UNICAP (CAP) and ADVIA Centaur (ADVIA). IgE-antibodies to species specific recombinant major allergens (SSMA) Api m1 for bee venom and Ves v5 for Vespula venom, were determined by ADVIA. 30 history and skin test negative patients served as controls . RESULTS: By CAP sensitivity was 1.0 for bee and 0.91 for Vespula venom, by ADVIA 0.99 for bee and 0.91 for Vespula venom. None of the controls were positive with either test. Double positivity was observed in 59% of allergic patients by CAP, in 32% by ADVIA. slgE to Api m1 was detected in 97% of bee and 17% of Vespula venom allergic patients, slgE to Ves v5 in 87% of Vespula and 17% of bee venom allergic patients. slgE to CCDs were present in 37% of all allergic patients and in 56% of those with double positivity and were more frequent in bee than in Vespula venom allergic patients . CONCLUSIONS: Double positivity of IgE to bee and Vespula venom is often caused by crossreactions, especially to CCDs. IgE to both Api m1 and Ves v5 indicates true double sensitization and immunotherapy with both venoms.

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[113] - Hofmann SC, Pfender N, Weckesser S, Huss-Marp J, Jakob T. Added value of IgE detection to rApi m 1 and rVes v 5 in patients with Hymenoptera venom allergy. J Allergy Clin Immunol. 2010 Aug 16. [Epub ahead of print]

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[114] - Mittermann I, Zidarn M, Silar M, Markovic-Housley Z, Aberer W, Korosec P et al. Recombinant allergen-based IgE testing to distinguish bee and wasp allergy. J Allergy Clin Immunol 2010;125:1300-1307

BACKGROUND: The identification of the disease-causing insect in venom allergy is often difficult. OBJECTIVE: To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. METHODS: Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). RESULTS: The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. CONCLUSION: Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy.

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[115] - Müller UR, Johansen N, Petersen AB, Fromberg-Nielsen J, Haeberli G. Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m 1 and Ves v 5. Allergy 2009;64:543-548

BACKGROUND: In patients with hymenoptera venom allergy diagnostic tests are often positive with honey bee and Vespula venom causing problems in selection of venoms for immunotherapy . METHODS: 100 patients each with allergic reactions to Vespula or honey bee stings and positive i.e. skin tests to the respective venom, were analysed for serum IgE to bee venom, Vespula venom and crossreacting carbohydrate determinants (CCDs) by UNICAP (CAP) and ADVIA Centaur (ADVIA). IgE-antibodies to species specific recombinant major allergens (SSMA) Api m1 for bee venom and Ves v5 for Vespula venom, were determined by ADVIA. 30 history and skin test negative patients served as controls . RESULTS: By CAP sensitivity was 1.0 for bee and 0.91 for Vespula venom, by ADVIA 0.99 for bee and 0.91 for Vespula venom. None of the controls were positive with either test. Double positivity was observed in 59% of allergic patients by CAP, in 32% by ADVIA. slgE to Api m1 was detected in 97% of bee and 17% of Vespula venom allergic patients, slgE to Ves v5 in 87% of Vespula and 17% of bee venom allergic patients. slgE to CCDs were present in 37% of all allergic patients and in 56% of those with double positivity and were more frequent in bee than in Vespula venom allergic patients . CONCLUSIONS: Double positivity of IgE to bee and Vespula venom is often caused by crossreactions, especially to CCDs. IgE to both Api m1 and Ves v5 indicates true double sensitization and immunotherapy with both venoms.

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[116] - Mahler V, Gutgesell C, Valenta R, Fuchs T. Natural rubber latex and hymenoptera venoms share Immunoglobin E-epitopes accounting for cross-reactive carbohydrate determinants. Clin Exp allergy 2006;36:1446-1456

BACKGROUND: Epidemiological data on the prevalence and risk factors of latex sensitization have suggested a significant association between latex sensitization and the presence of one or more positive skin prick test responses to aeroallergens, food allergens and to one or more insect venoms. Xylose and core 3-fucose are typical complex glycans in plants and are foreign to mammals. Plant N-glycans and insect N-glycans may cross-react in humans . OBJECTIVE: The aim of our study was to investigate whether there are cross-reactive IgE-binding structures in natural rubber latex (NRL) and hymenoptera venoms and to examine their nature . METHODS: Hundred and twenty-five consecutive patients with insect venom allergy were screened for coincidental latex-specific IgE. IgE-binding components in the venoms from Apis mellifera and/or vespula species and in NRL extracts were characterized by IgE-immunoblotting to the natural allergen sources and determination of specific IgE to recombinant allergens. Cross-reactive components were investigated by inhibition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE-epitopes was further examined by specific IgE-binding to cross-reactive carbohydrate determinants (CCD) in bromelain and horseradish peroxidase as well as by periodate treatment . RESULTS: NRL glove extracts inhibited patients' serum IgE-binding to venom allergens. Vice versa, the IgE-binding to latex glove extracts could be inhibited by pre-incubation with the insect venoms. Specific IgE-binding to recombinant latex allergens was absent, whereas the cross-reactive IgE-epitopes were sensitive to periodate treatment and specific IgE to CCD (MMXF and MUXF type) could be detected . CONCLUSION: Insect venoms and NRL share IgE-binding CCD that may be responsible for positive serological test results to NRL in patients with insect venom allergy. This copositivity occurs frequently (13.6%) among venom-allergic individuals and did not elicit clinical symptoms upon contact to latex in the patients examined. In contrast, true cosensitization to insect venoms and NRL allergens can occur and may not be missed.

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[117] - Jappe U, Hoffmann M, Burow G. Potential screening allergens for detection of carbohydrates as cause for in vitro double positivity to honeybee and yellow jacket venom as well as rubber latex in patients with stinging insect allergy. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°69

IgE-double positivity for honeybee (HB) and yellow jacket (YJ) venom sometimes causes diagnostic difficulties concerning therapeutical strategies. True double sensitisation may account for some of the results. Another explanation is crossreactivity caused by sequence homologies between the venom hyaluronidases. However, previous investigations revealed that polyreactive human sera binding through carbohydrates with HB venom also bind to a broad range of pollen and food allergens, and, according to few reports, to rubber latex as well. In this study, 98 patients, who attended the clinic between 1996-2003 with stinging insect allergy and additionally were CAP RAST positive for both, HB and YJ IgE, were investigated for carbohydrate-binding IgE. The following allergens were used in the CAP-RAST: HB, YJ, whole extracts of timothy pollen, rape pollen, and rubber latex, bromelain, and horse radish peroxidase, all carrying glycans, except two additional allergens, the recombinant timothy pollen components Phl p1 and Phl p5, which were produced by expression in E. coli without a glycosylation step. 26/98 patients with CAP RAST-positivity to both venoms did not show IgE-positivity to other allergens, 72/98 additionally reacted to carbohydrate-carrying allergens. 52 out of these did not detect the recombinant components Phl p1 and Phl p5, and 20/72 bound with the whole range of allergens. Out of 72 carbohydrate-reactive sera, 20 were binding to rubber latex. For 20 sera, an additional reciprocal inhibition assay was performed with HB and YJ venom, respectively. 16/20 were negative for carbohydrate-binding IgE, 6/16 showed a homologous but not heterologous reaction, indicating a true double sensitisation of those patients. In cases of IgE double positivity to both insect venoms supplementary CAP RAST with at least one carbohydrate containing allergen plus recombinant allergens, e.g. timothy pollen allergen plus its recombinant components and/or horse radish peroxidase should be performed. The detection of carbohydrate-binding IgE as well as a subsequent reciprocal inhibition assay are essential diagnostic tools to specify initial CAP RAST results.

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[118] - Jappe U, Hoffmann M, Burow G, Enk A. Pitfalls of in vitro-allergy diagnostic: glycan-associated epitope sharing of insect venom and natural rubber latex allergens. Significance of screening allergens, reciprocal inhibition, and recombinant allergens. Allergy Clin Immunol Int 2005;17(Suppl. 1):191-192

Background: IgE-double positivity for honeybee (HB) and yellow jacket (YJ) venom may be due to true double sensitization, artefacts caused by increased sensitivity of the test system and cross-reactive carbohydrate determinants (CCD). It may cause diagnostic difficulties concerning therapeutical strategies. Previous investigations revealed that IgE-positive human sera for both, HB and YJ venom, also bind to a broad range of plant allergens due to CCD. Sera of patients with insect venom allergy who had attended the department between 1995 and 2004 were evaluated. Methods: 146 patients with suspected stinging insect allergy and CAP FEIA-double positivity were investigated for specific (s)IgE to additional CCDcontaining allergens: whole extracts of timothy pollen, rape pollen, rubber latex, bromelain, and horse radish peroxidase (HRP). Sera positive for rubber latex IgE were further investigated with the recombinant latex components rHev b1, b2, b3, b5, b6.01, b6.02, b8, b9, and b11. The corresponding patients were further investigated for clinical relevance of the CAP-FEIA-results (questionnaire, skin prick test). Reciprocal inhibition assays with both venoms (100µg/ml) and HRP (500µg/ml) were performed. Results: 38/146 patients were sIgE-positive to both venoms only. 108/146 additionally reacted to CCD-carrying allergens. 91/108 CCD-reactive sera had sIgE to rubber latex. 37/56 rubber latex-IgE-positive sera were negative for anti-recombinant-latex-IgE, 19/56 were positive for at least one recombinant component. 21/56 of the corresponding patients who were already available for a skin prick test with natural rubber latex and a questionnaire were negative in history and skin prick test. An additional reciprocal inhibition assay was performed for 29 sera with HB and YJ venom, respectively. 8/29 showed a homologous but not heterologous reaction, indicating a true double sensitization of those patients. Inhibition with HRP in 24 sera revealed 100% inhibition of anti-HRP-IgE binding, in 3/24 HRP completely inhibited IgE-binding to YJ, and in 11/24 sera to HB. Conclusions: In cases of IgE-positivity to both insect venoms supplementary CAP FEIA with at least one CCD containing allergen as screening test should be performed. Subsequent reciprocal inhibition is an essential diagnostic tool to specify cross-reacting CAP FEIA results as will most probably be recombinant allergens after having been evaluated for their specificity and clinical relevance.

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[119] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[120] - Jappe U, Hoffmann M, Huebsch-Mueller C, Enk A, Raulf-Heimsoth M. Specific IgE-antibody reaction to cross-reactive carbohydrate determinants (CCD) in hymenoptera venoms: comparison of two different in vitro-tests. Allergy 2007;62(suppl. 83):50

Background: IgE-positivity for both hymenoptera venoms is mostly due to cross-reactive carbohydrate determinants (CCD). This study evaluated CCD-IgE-binding intensity in two in vitro-IgE-detection-methods. Patients and methods: 56 patients with stinging insect allergy and anti-CCD-sIgE were investigated with further CCD-allergens: timothy grass (Phl p), natural rubber latex (NRL), bromelain (BRO), and horse radish peroxidase (HRP) in 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (DPC Biermann, Germany), based on liquid phase technology. The new CCD-ImmunoCAP, a MUXF3-type carbohydrate and ascorbate oxidase (DPC), also a glykan-containing allergen, were used as well. The sera were further investigated via hymenoptera venom- immunoblot (DPC). Results: All sera had sIgE to both insect venoms. In CAP FEIA, 47/56 (83.9%) had sIgE to NRL, 52/56 (92.9%) to Phl p, 50/56 (89.2%) to BRO, 50/56 (89.2%) to HRP and 48/56 (85.7%) to CCD-CAP. In Immulite, 30/56 (53.6%) had sIgE to NRL, 40/56 (71.4%) to BRO, 46/56 (82.1%) to HRP, 40/56 (71.4%) to Phl p, and 45/56 (80.4%) to ascorbate oxidase. In CAP FEIA sIgE-concentrations to BRO and HRP, showed a strong Pearson correlation with 0.9994 (p<0.0001). Although they differed individually in Immulite, the Pearson correlation was still good (0.6953; p=0.01). However, via Immulite, HRP-sIgE could be measured in 8 subjects, where no BRO-sIgE were detected, whereas only 2 individuals had sIgE to BRO and none to HRP. Regarding the class-grading, the differences between BRO and HRP in Immulite measured were 3 classes in 3, 2 classes in 20, and 1 class in 16 subjects. This phenomenon is dependent on BRO-DPC and is mirrored by a non-significant Pearson correlation for BRO-DPC/BRO-CAP (0.488; p=0.56), BRO-DPC/HRP-CAP (0.497; p=0.51) and a significant Pearson correlation for HRP-DPC/BRO-CAP (0.8905; p<0.0001). In immunoblot, all sera reacted with the glycosylated allergens. As in CAP FEIA the IgE-binding to the CCD structures was detected in more sera and was comparable with all the tested CCD allergens, it might be assumed that these epitopes are equally presented on the different ImmunoCAPs. The differences in the IgE-binding to BRO and HRP in the Immulite E 2000 system might be due to their presentation in these allergens in liquid phase. However, HRP seems to be the favourable screening allergen in cases of IgE-double-positivity in insect venom allergic patients.

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

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

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[122] - Sabbah A, Hassoun S, Drouet M, Lauret MG, Doucet M. Le syndrome guêpe/moustique. Allerg Immunol (Paris) 1999;31:175-184

The authors describe for the first time the Wasp/Mosquito syndrome. The allergenic cross-reactivity between Vespula wasp venom and mosquito extract and the reverse rest objectively on several criteria. The frequency of the association between sensibilization to Vespula wasp venom and mosquito: In 10 subjects who had specific IgE to Vespula wasp venom 3 also had mosquito-specific IgE (30%), but out of 11 subjects with positive mosquito-specific IgE 10 also were positive with Vespula wasp venom specific IgE (about 91%). Three observations were documented showing a clinical and biological relationship between sensitisation to mosquito and wasp venom. A first patient had an anaphylactic reaction after a dozen mosquito bites. The allergy assessment confirmed sensitisation to the mosquitos, but also to Vespides (wasp, hornet) venoms. A second patient had an anaphylactic reaction after a wasp sting in 1997. From that date, he had local reactions, indeed loco-regional, to mosquito bites. The allergy assessment confirmed the double sensibilization to mosquito and wasp venom. Finally, a third patient at first had local reactions, also loco-regional, to mosquito bites, then two systemic reactions after mosquito bites. A first allergy assessment, made exclusively to hymenoptera venoms (not to mosquitos), showed a sensitivity to wasp venom and the patient, by mistake, was desensitised to wasp venom, which did not prevent recurrence after mosquito bites. After a second time, the allergy assessment for hymenoptera and mosquito, confirmed a double sensitisation (mosquito and Vespula wasp). Indirect criteria such as parallel increase in specific IgEs to Vespula wasp venom and mosquito in a patient who was desensitised only to mosquito and who had not been re-stung by wasps. Finally on direct criteria, such as: The inhibition technique by CAP RAST, which was positive in two ways: Vespula wasp--specific IgE was inhibited by mosquito extract, and vice versa. Electrophoresis, which showed a protein common to Vespula wasp venom and mosquito extract of M.W. of the order or 42KD and isoelectric point between 4.5 and 5. An immunoblot study identified this common protein with a M.W. of 44KD which is the same as hyaluronidase.

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[123] - Hassoun S, Drouet M, Sabbah A. Anaphylaxie au moustique: à propos de 2 cas cliniques. Allerg Immunol (Paris) 1999;31:285-287

Allergy to mosquitoes that produces anaphylaxis is very exceptional. We present two observations of anaphylaxis to mosquitoes with good correlation between the clinical history and the immunological indications of which the basophil activation test proved to be effective. The many allergens of mosquitoes are contained in the juices and salivary glands and their molecular weights are between 22 and 95 kD. Specific immunotherapy, which uses whole body extracts of mosquito, gives good results of protection that induces excellent tolerance and has very good efficacy.

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[124] - Quercia O, Emiliani F, Foschi FG, Stefanini GF. The wasp-horsefly syndrome. Eur Ann Allergy Clin Immunol 2008;40:61-63

Here are two cases of two male patients of 57 and 62 years of age, already known as allergic to stinging hymenoptera venom, who after a horsefly bite have presented a serious 3-4 degree-type Mueller classification systemic reaction. The diagnosis has been carried out clinically and after an accurate environmental anamnesis and along with prick tests and RAST, further specific entomological confirm. In literature the so called wasp-mosquito-syndrome has been indicated where hyaluronidase has been referred to as the cross allergen, between the hymenoptera venom and the mosquito saliva, which likely triggers the reaction. We believe that it is also possible to take into consideration a wasp-horsefly-syndrome as well, supposing the increased risk of anaphylactic reactions to Tabanidae bites, relatively frequent in areas with animals and streams, in subjects sensitized to stinging hymenoptera. We also suggest the possibility that in these subjects some systemic reactions are due in fact to Tabanidae bites and not so much for the failure of a possible active ITS of stinging hymenoptera.

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[125] - Sabbah A, Hassoun S, Drouet M, Lauret MG, Doucet M. Le syndrome guêpe/moustique: extension de l'allergénicité croisée au taon. Allerg Immunol (Paris) 2000;32:16-19

The crossed allergenicity between wasp venom and mosquito extract was shown during recent work based on clinical observations and correlation studies between different biological parameters, indicating an IgE-dependent biological mechanism. A common protein was identified by Immunoblot. From observation of one of our patients involved in this work, we examined the possibility of the extension of crossed reactivity between wasp, mosquito and horsefly. In effect, our patient presented an anaphylactic reaction with neurological complications from attack on the central grey nucleus, shown by IRM; the immunological study showed a common protein between wasp venom and the total extracts of mosquito and horsefly.

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[126] - Hassoun S, Drouet M, Sabbah A. Anaphylaxie au moustique: à propos de 2 cas cliniques. Allerg Immunol (Paris) 1999;31:285-287

Allergy to mosquitoes that produces anaphylaxis is very exceptional. We present two observations of anaphylaxis to mosquitoes with good correlation between the clinical history and the immunological indications of which the basophil activation test proved to be effective. The many allergens of mosquitoes are contained in the juices and salivary glands and their molecular weights are between 22 and 95 kD. Specific immunotherapy, which uses whole body extracts of mosquito, gives good results of protection that induces excellent tolerance and has very good efficacy.

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

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

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	Abeilles	Bourdons	Guêpes		Frelons
			Polistes	Vespula	Dolichovespula	Vespa
Phospholipases A2	X	X
Phospholipases A1			X	X	X	X
Hyaluronidases	X	X	X	X	X	X
Phosphatases acides	X	X
Sérine protéases	X	X	X
Ag5			X	X	X	X

Abeilles, guêpes, frelons : classification

Les allergènes des Apides et des Vespides

Diagnostic d’allergie aux Hyménoptères

Immunothérapie spécifique

Doubles positivités

Bilan diagnostique négatif malgré une histoire clinique positive

Hyménoptères et CCD

L’apport des allergènes recombinants dans les doubles positivités

Venins d’hyménoptères et latex

Co-réactivités hyménoptères-diptères

Rubriques

Dans la même rubrique

L’allergie au latex