Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis.

Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one-dimensional gel electrophoresis (D1-DE) as an alternative to the 2-DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one-dimensional combination of IEF and SDS-PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43 kDa. D1-DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one-dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin-based protein-protein interactions. Therefore, D1-DE could be used in routine work as a convenient alternative to 2-DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.

Immediate Hypersensitivity to Contrast Agents: The French 5-year CIRTACI Study.

BACKGROUND: Iodinated and gadolinium-based contrast media (ICM; GBCM) induce immediate hypersensitivity (IH) reactions. Differentiating allergic from non-allergic IH is crucial; allergy contraindicates the culprit agent for life. We studied frequency of allergic IH among ICM or GBCM reactors. METHODS: Patients were recruited in 31 hospitals between 2005 and 2009. Clinical symptoms, plasma histamine and tryptase concentrations and skin tests were recorded. Allergic IH was diagnosed by intradermal tests (IDT) with the culprit CM diluted 1:10, "potentially allergic" IH by positive IDT with pure CM, and non-allergic IH by negative IDT. FINDINGS: Among 245 skin-tested patients (ICM = 209; GBCM = 36), allergic IH to ICM was identified in 41 (19.6%) and to GBCM in 10 (27.8%). Skin cross-reactivity was observed in 11 patients with ICM (26.8%) and 5 with GBCM (50%). Allergy frequency increased with clinical severity and histamine and tryptase concentrations (p < 0.0001). Cardiovascular signs were strongly associated with allergy. Non-allergic IH was observed in 152 patients (62%) (ICM:134; GBCM:18). Severity grade was lower (p < 0.0001) and reaction delay longer (11.6 vs 5.6 min; p < 0.001). Potentially allergic IH was diagnosed in 42 patients (17.1%) (ICM:34; GBCM:8). The delay, severity grade, and mediator release were intermediate between the two other groups. INTERPRETATION: Allergic IH accounted for < 10% of cutaneous reactions, and > 50% of life-threatening ones. GBCM and ICM triggered comparable IH reactions in frequency and severity. Cross-reactivity was frequent, especially for GBCM. We propose considering skin testing with pure contrast agent, as it is more sensitive than the usual 1:10 dilution criteria.

[Immunoproteomics of non water-soluble allergens from 4 legumes flours: peanut, soybean, sesame and lentil]. / Analyse immunoprotéomique des allergènes non-hydrosolubles de farines de quatre légumineuses : arachide, soja, sésame et lentille.

Peanut, soybean, sesame and lentil are members of legumes worldwide consumed by human that can induce food allergy in genetically predisposed individuals. Several protein allergens, mainly water-soluble, have been described. We studied the non water-soluble fraction from these 4 food sources using immunoproteomics tools and techniques. Flour extracts were solubilized in detergent and chaotropes and analysed in 1 and 2 dimensional gel electrophoresis (2D). Results showed numerous proteins exhibiting wide ranges of isoelectric points and relative molecular masses. When IgE immunoreactivities of 18 food allergy patients were individually tested in 1 and 2D western-blots, a very diversified IgE repertoire was observed, reflecting extensive cross-reactivities but also co-sensitizations. Besides already well known and characterized allergens, mass spectrometry analysis allowed the identification of 22 allergens undescribed until now: 10 in peanut, 2 in soybean, 3 in sesame and 7 in lentil. Three allergens are legume storage proteins and the others belong to transport proteins, nucleotide binding proteins and proteins involved in the regulation of metabolism. Seven proteins are potentially similar to allergens described in plants and fungi and 11 are not related to any known allergen. Our results contribute to increase the repertoire of legume allergens that may improve the diagnosis, categorize patients and thus provide a better treatment of patients.

Immediate allergic hypersensitivity to quinolones associates with neuromuscular blocking agent sensitization.

BACKGROUND: We identified a case of quinolone allergic hypersensitivity associated with quaternary ammonium (QA) sensitization, the allergic determinant of neuromuscular blocking agents (NMBAs). Concomitant sensitization to several chemically different drugs is rarely reported and raises the question of a nonfortuitous association. OBJECTIVE: We evaluated a potential association between quinolone immediate allergic hypersensitivity and NMBA sensitization. METHODS: QA-specific IgE detection was prospectively performed in 26 patients who presented an immediate hypersensitivity reaction to quinolones: 17 with a confirmed allergic hypersensitivity and 9 with allergic hypersensitivity not confirmed. We also included a control population of 88 outpatients without a history of quinolone or NMBA hypersensitivity. Patients with positive QA-specific IgE benefited from a NMBA allergologic workup. RESULTS: The prevalence of positive QA-specific IgE was significantly higher in patients with quinolone allergic hypersensitivity (9/17, 53%) compared with patients with allergic hypersensitivity not confirmed (1/9, 11%) than in controls (3/88, 3.4%). In the quinolone allergic population, ofloxacin elicited inhibition of the 4 positive QA-specific IgE sera tested, in a dose-response manner. Among the 9 patients with positive QA-specific IgE, the QA sensitization (positivity of specific IgE) was confirmed by positive skin tests and/or basophil activation tests to at least 1 NMBA in 5 of the 7 tested patients. CONCLUSION: We report here the first documentation of a high prevalence of QA sensitization in patients with quinolone allergic hypersensitivity. These results suggest a new way for NMBA sensitization. It thus seems appropriate to investigate NMBA sensitization when quinolone allergic hypersensitivity is diagnosed.

Differential IgE sensitization to cypress pollen associated to a basic allergen of 14 kDa.

The common cypress (Cupressus sempervirens) (Cups) pollen represents the first cause of respiratory allergies in the Mediterranean basin. The aim of this study was to characterize a novel 14-kDa cypress pollen allergen (BP14) allowing a clear dissociation of IgE sensitization patterns among allergic patients. The biochemical and immunochemical characterization of BP14 included determination of its isoelectric point, molecular mass, extraction kinetics, IgE binding prevalence, the presence of bromelain-type cross-reactive carbohydrate determinant and its IgE reactivity under reducing conditions. The presence of potential cross-reactive homologues in closely related cypress species, i.e. Cupressus arizonica (Cupa) and Cryptomeria japonica (Cryj), as well as in several taxonomically unrelated species was also investigated. According to our results, BP14 is easily and quickly solubilized in phosphate-buffered saline and exhibits several allergenic isoforms covering a broad range of pI (6.5-10.5). This allergen displays heat-stable conformational epitopes and does not include cross-reactive carbohydrate determinants, in contrast to high molecular weight cypress allergens. BP14 is expressed at higher levels in Cups than in Cupa and Cryj. No IgE cross-reactivity was found between the 14-kDa Cups pollen protein and proteins from some other non-Cupressaceae pollen allergenic sources such as orchard, timothy, wheat, maize, birch, ash and pine. Thus, IgE reactivity to BP14 is specific to Cupressaceae and discriminates two groups of patients allergic to cypress pollen. It might correspond to a relevant marker in relation to the sensitization process and/or the symptoms observed in some cypress-pollen-allergic patients. Furthermore, the description of BP14 should improve the diagnosis of cypress pollinosis.