Showing papers by "Domingo Barber published in 2012"

Component‐resolved diagnosis of vespid venom‐allergic individuals: phospholipases and antigen 5s are necessary to identify Vespula or Polistes sensitization

Abstract: Background Cross-reactivity between hymenoptera species varies according to the different allergenic components of the venom. The true source of sensitization must therefore be established to ensure the efficacy of venom immunotherapy. Objective In the Mediterranean region, Polistes dominulus and Vespula spp. are clinically relevant cohabitating wasps. A panel of major vespid venom allergens was used to investigate whether serum-specific IgE (sIgE) could be used to distinguish sensitization to either vespid. Methods Fifty-nine individuals with allergic reactions to vespid stings and positive ImmunoCAP and/or intradermal tests to vespid venoms were studied. sIgE against recombinant and natural venom components from each wasp species was determined using the ADVIA Centaur® system. Results sIgE against recombinant antigen 5s sensitization to be detected in 52% of the patients tested (13/25). The sensitivity increased to 80% (20/25), when using natural antigen 5s, and to 100% with the complete panel of purified natural components, because the sIgE was positive to either the antigen 5s (Pol d 5/Ves v 5) or to the phospholipases (Pol d 1/Ves v 1) of the two vespids, or to both components at the same time. In 69% of cases, it was possible to define the most probable sensitizing insect, and in the rest, possible double sensitization could not be excluded. Vespula hyaluronidase was shown to have no additional value as regards the specificity of the assay. Conclusions The major allergens of P. dominulus’ and Vespula vulgaris’ venom, namely phoshpholipases and antigen 5s, are required to discriminate the probable sensitizing species in vespid-allergic patients.

Analysis of mite allergic patients in a diverse territory by improved diagnostic tools.

Abstract: Summary Background There are few studies comparing the sensitization with mite allergens from different mite species which could potentially be the cause of allergy. Objective To improve the diagnosis of mite allergic patients from a diverse territory in which D. pteronyssinus/D. farinae mites together with storage mites could be present in the environment. Methods Four hundred and seventy-seven patients (both children and adults) from different regions, covering the main mite prevalent areas of Spain, were recruited. sIgE to eight allergens was measured together with SPT to whole mite extracts, level of mite allergen exposure, and specific IgG4. BAT and CAST was performed in a subgroup of patients. Results D. pteronyssinus and L. destructor were more prevalent in Atlantic areas, whereas D. farinae predominate in Mediterranean areas. About 90% of patients were sensitized to group 1 and/or group 2 allergens. Group 2 was the most prevalent, and the IgE response/ intensity of sensitization in BAT was higher. sIgE to Der p 2/Der f 2 was almost fully cross-reactive, but no cross-reactivity was detected with Lep d 2. Group 1 allergens were also cross-reactive, but in some patients a species-specific response was observed. sIgE to Lep d 2 was associated with SPT results to storage mites. Sensitization to Der p 1 was more frequent in children, whereas Lep d 2 sensitization was more frequent in adults. A higher ratio IgE/IgG4 to Der p 2 was associated with the presence of allergic asthma. Conclusion An improved diagnosis algorithm has been established. Group 2 allergens seem to have a leading role in mite allergy, but as group 1 sensitization could be speciesspecific in some patients and its prevalence is higher in children, an adequate balance on major mite species and major allergens must be consider in the design of mite allergy

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

Abstract: The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

Abstract: Background Treatment with aqueous and aluminum hydroxide (Al[OH] 3 )–adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. Objective The aim of the present study was to assess these purified HBV immunotherapy preparations in situ . Methods Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. Results Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH) 3 -adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. Conclusion The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH) 3 -adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

Can component-resolved diagnosis overturn the current knowledge on vespid allergy?

Abstract: To the Editor In the study by Monsalve et al. (1), 45 individuals with allergic reactions to vespid stings and positive tests to vespid venoms were studied by measuring their IgE to single recombinant and natural allergens from Vespula and Polistes. The results showed that such component-resolved diagnosis (CRD) allowed to recognize the really causative venom in 31 cases (69%), while in 14, a double sensitization was apparent. However, among the patients identified by CRD as monosensitized, 22 (71%) were sensitized to Polistes venom and 9 (29%) were sensitized to Vespula venom. This is in striking contrast with what we know about entomological and clinical aspects of hypersensitivity to vespids. In fact, Vespula spp. is much more aggressive than Polistes spp., this making the former the culprit insect in most cases of allergic reactions to vespid venom (2). In the first study demonstrating by the RAST-inhibition technique that in most patients with double sensitization to Vespula and Polistes only one venom was really causative, Polistes venom was identified only in 4.3% of patients (3). One may consider that the geographic distribution of Polistes is especially relevant in Mediterranean areas including Spain (4), but in an epidemiologic study conducted in Spain, the percentage of subjects with a positive RAST for Vespula venom was more than double the percentage found for Polistes (5). The most likely explanation of the finding from Monsalve et al. is provided by the low number of patients and thus on the stochastic nature of the data. Had the authors examined a number of patients as predetermined by statistical calculation, the results would have been different. However, any other explanation from the authors will be useful to appraise the real role of CRD in Hymenoptera venom allergy.