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ABSTRACT: Although Cupressus sempervirens (Cups) pollen represents one of the main aeroallergens in southern Europe, only two Cups allergens have yet been identified and reported: Cup s 1 and Cup s 3. The aim of this study was to identify allergens in cypress pollen using an immuno-proteomic approach. A sequential pollen protein extraction was developed and supplemented by a combinatorial peptide ligand library (CPLL) treatment to select low-abundance species. Control extracts and CPLL eluates have then been resolved by 1-DE and 2-DE gel electrophoresis, blotted and confronted with sera from cypress allergic patients. Extracted proteins including IgE-binding components were identified using nanoLC-MS/MS analysis. A total of 108 unique gene products were identified analyzing the eluates and control loaded onto 1-DE SDS-PAGE. Forty proteins were identified in control samples and 68 supplementary species upon CPLL treatment. Out of the 12 IgE-binding proteins characterized in 2-DE gels, 9 were already reported as allergens in various sources including the two major known allergens of Cupressaceae (groups 1 and 2). Three IgE-binding proteins, not previously reported as allergens, are newly described. The improvement in protein extraction combined with the enrichment of low-abundance species allowed us to extend the repertoire of potential cypress pollen allergens.
Journal of proteomics 07/2012; · 5.07 Impact Factor
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ABSTRACT: 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.
FEBS Journal 02/2012; 279(8):1445-55. · 3.79 Impact Factor
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ABSTRACT: 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.
Electrophoresis 02/2012; 33(3):462-9. · 3.30 Impact Factor
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ABSTRACT: Grass pollen is one of the most important vectors of aeroallergens. Under atmospheric conditions, pollen grains can release pollen cytoplasmic granules (PCGs). The allergens associated with these intrinsic subfractions induce, in laboratory animals as well as in asthmatic patients, allergic and inflammatory responses. The objectives of this study were to characterize the PCGs' intrinsic allergens and to compare them with those of pollen grains. The water-soluble proteins were extracted from pollen grains and their PCGs. IgE-binding proteins were analyzed and characterized through an allergomic strategy: 1- and 2-dimensional gel electrophoresis (1-DE and 2-DE), immunoblotting, using grass-pollen-sensitized patient sera, mass spectrometry (MS) analysis, and database searching. Several of the allergens listed in the IUIS nomenclature, Phl p 1, 4, 5, 6, and 12, were detected in pollen and PCG extracts, whereas Phl p 11 was found only in PCGs, and Phl p 2 as well as Phl p 13 were found only in pollen extract. Some other allergens not listed in the IUIS nomenclature were also characterized in both pollen and PCG extracts. Since the major grass pollen allergens were found in PCGs and because of their small size, these submicronic particles should be considered as very potent sensitizing and challenging respirable vectors of allergens.
Journal of Proteome Research 12/2011; 11(2):1208-16. · 5.11 Impact Factor
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ABSTRACT: Grass pollen is one of the most important aeroallergens in Europe. It highly contributes to respiratory allergic diseases, mainly allergic rhinitis. In contact to water or airborne pollutants, pollen grains can release pollen cytoplasmic granules (PCGs) containing allergens. Because of their size (<5 μm), PCGs may penetrate deeper into the lungs to induce higher allergic responses, such as asthma. They have been associated with thunderstorm-related asthma. The aim of this study was to evaluate, with Brown Norway rats, the allergenic potential of isolated PCGs and to compare it with the allergenicity of whole timothy grass pollen.
Rats were sensitized (day 0) and challenged (day 21), in controlled comparative conditions, with pollen grains (0.5 mg) or PCGs (4.5 × 10⁷ and 0.5 mg). At day 25, blood samples, bronchoalveolar lavage fluid (BALF) and bronchial lymph node were collected. IgE and IgG1 levels in sera were assessed by ELISA. Alveolar cells, protein and cytokine concentrations were quantified in BALF. T cell proliferation, in response to pollen or granules, was performed by lymph node assay.
The results showed that proliferative responses of lymph node cells were similar in PCG- and pollen-sensitized rats. IgE and IgG1 levels were higher in pollen- than in PCG-sensitized rats. However, eosinophils, lymphocytes and pro-allergy cytokines in BALF were higher in PCG- than in pollen-sensitized rats.
Thus, PCGs, able to deeply penetrate in the respiratory tract, induced local and strong allergic and inflammatory responses more linked with asthma- than rhinitis-related allergic symptoms.
International Archives of Allergy and Immunology 01/2011; 154(2):128-36. · 2.40 Impact Factor
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ABSTRACT: : Grass pollen grain, an important aeroallergen, can disperse in the environment pollen cytoplasmic granules (PCGs) able to release water-soluble allergens when they are washed out by rainfall. The allergenicity of these washed PCGs is, however, preserved.
: The purpose of the study was to assess the allergenic potential of washed and unwashed PCGs, from Phleum pratense pollen grains, in the Brown Norway rat, and to study the IgE reactivity of sera of sensitized rats to water-soluble and water-insoluble extracts of PCGs and pollen grains.
: Rats were sensitized and challenged intratracheally with washed or unwashed PCGs or pollen grains. Using water-soluble and -insoluble extracts of pollen grains and/or PCGs, IgE ELISA and immunoblotting were performed with rat sera. Proliferation of bronchial lymph node cells was monitored by [H]-thymidine incorporation in a lymph node assay. Alveolar cells, proteins, and TH1 and TH2 cytokines were quantified in bronchoalveolar lavage fluid.
: Rats sensitized with unwashed PCGs showed a predominant humoral response with high serum IgE and reactivity to water-soluble and -insoluble proteins together with low lymph node cell proliferation. Conversely, in rats sensitized to washed PCGs, cellular responses were higher with significant increases in eosinophils, lymphocytes, and TH2 cytokines observed in bronchoalveolar lavage fluid.
: Allergic and inflammatory responses were induced by both grass pollen grains and their isolated washed and unwashed PCGs. However, on the basis of humoral and cellular responses, differential patterns were observed. Water-insoluble allergens seem to play a role in the centrally mediated inflammatory response, whereas water-soluble allergens may be involved in the peripheral humoral response.
World Allergy Organization Journal 01/2011; 4(1):4-12.
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ABSTRACT: : Cypress pollen is becoming an increasing cause of respiratory allergy in some regions worldwide.
: The aim of this study was to determine some of the main allergens implicated in the common cypress (C. sempervirens) pollen allergy.
: Pollen extracts were optimized by using some detergents and chaotropes in order to solubilize both water and non-water soluble proteins. C. sempervirens pollen extracts were resolved by one and two dimensional electrophoresis and assayed with sera of allergic subjects.
: Five predominant allergens with apparent molecular masses ranging from 14 to 94 kDa were detected. Two principal IgE-binding patterns were clearly distinguishable: a first one represents patients with a heterogeneous IgE reactivity to several allergens (pI 3.5-8.5) with molecular masses ranging from 35 to 94 kDa (HMW). The second one corresponds to little less than 50 percent of tested patients with specific IgE binding to 2-3 spots (pI 10-11) of about 14 kDa and weak or no reactivity to HMW allergens.
: The extraction of water insoluble proteins allows the revelation of novel allergens as well as different allergen sensitization patterns in the C. sempervirens pollen allergy. These novel IgE reactive components may subsequently be applied to expand the panel of well-defined cypress pollen molecules for a more efficient allergen-based diagnosis and therapy.
World Allergy Organization Journal 08/2010; 3(8):229-34.
