Hélène Sénéchal

In-depth exploration of Hevea brasiliensis latex proteome and "hidden allergens" via combinatorial peptide ligand libraries.

The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81 kDa), proteasome subunit (30 kDa), protease inhibitor (8 kDa), hevamine A (43 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.

Modifications of Phleum pratense Grass Pollen Allergens following Artificial Exposure to Gaseous Air Pollutants (O3, NO2, SO2)

Background: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. Methods: Timothy grass pollen was exposed to ozone (O3), nitrogen dioxide (NO2) and sulphur dioxide (SO2) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. Results: For O3-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO2 exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO2 treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO2/O3 or NO2/SO2 showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O3 acidification was also observed with the NO2/O3 mix. Conclusion: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.

Mapping of Dermatophagoides farinae mite allergens by two-dimensional immunoblotting

BACKGROUND Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized. OBJECTIVE This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis. METHODS D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. RESULTS 2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus. CONCLUSION 2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.

Genetics and specific immune response in allergy to birch pollen and food: Evidence of a strong, positive association between atopy and the HLA class II allele HLA-DR7

BACKGROUND In some geographic areas birch pollen represents the most prominent cause for airborne allergic diseases. Up to 70% of patients allergic to birch pollen are hypersensitive to fruits, especially apples. Associations have been found, in some instances, with a sensitivity to aeroallergens and HLA class II genes. OBJECTIVES We investigated whether susceptibility or resistance to birch pollen allergy with and without food allergy was associated with HLA class II genes. METHODS Blood samples were obtained from 2 groups of unrelated European-born white adults: 42 atopic patients (31 of them with asthma) and 42 healthy control subjects with no personal or familial history of asthma or atopy. Their antibody responses to birch pollen, apples, grass, and weed pollens were evaluated by skin tests, RASTs, and immunoprints. Genomic DNA was extracted from PBLs. The exons of DQA1, DQB1, DRB1, and DPB1 genes were selectively amplified by using the PCR method. Genotyping was carried out by digestion of the amplified DNA products with allele-specific endonucleases (PCR-RFLP), which recognize allelic variations in the polymorphic exon. RESULTS We found no significant differences in the frequency of DPB1 alleles between patients and control subjects. HLA class II DR4 and/or DR7 alleles were present in 42.6% of the patients and in only 2.4% of the healthy subjects. These results confirm a previous study of a group of polysensitized atopic patients, which showed that DR4 and DR7 alleles were rare in healthy control subjects and frequently observed in atopic subjects with or without concomitant asthma. CONCLUSION We conclude that the allele HLA-DR7 is significantly involved in the presentation of apple and pollen allergens. However, we suggest that this susceptibility is more related to atopy than to specific responses to allergens.

Allergomic study of cypress pollen via combinatorial peptide ligand libraries

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.

A Review of the Effects of Major Atmospheric Pollutants on Pollen Grains, Pollen Content, and Allergenicity.

This review summarizes the available data related to the effects of air pollution on pollen grains from different plant species. Several studies carried out either on in situ harvested pollen or on pollen exposed in different places more or less polluted are presented and discussed. The different experimental procedures used to monitor the impact of pollution on pollen grains and on various produced external or internal subparticles are listed. Physicochemical and biological effects of artificial pollution (gaseous and particulate) on pollen from different plants, in different laboratory conditions, are considered. The effects of polluted pollen grains, subparticles, and derived aeroallergens in animal models, in in vitro cell culture, on healthy human and allergic patients are described. Combined effects of atmospheric pollutants and pollen grains-derived biological material on allergic population are specifically discussed. Within the notion of “polluen,” some methodological biases are underlined and research tracks in this field are proposed.

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.

Isoelectric focusing in an ordered micropillar array.

An original micropillar array dedicated to electrophoretic separations has been developed. It consists of a rectangular zone of PDMS micropillars protruding on a PDMS block. This area has been chosen to mimic a diluted gel structure and remains uncovered to keep the ability to perform an immunoblot after the protein separation for further applications in the field of allergy diagnosis. The micropillar array geometry has been optimized by evaluating the influence of pillar shape, pillar size and interpillar distance on evaporation and IEF separation. The separation conditions namely electrolyte composition, temperature and sample loading have been studied. Finally a protein mixture with pI ranging from 4.7 to 10.6 has been successfully separated within this microdevice by IEF without decreasing the resolving power obtained with conventional minigel. The micropillar array developed for electrophoretic separations leads to much shorter analysis times and can be reused several times while gels are disposable.

IgE Reactivity to Common Cypress (C. Sempervirens) Pollen Extracts: Evidence for Novel Allergens

BackgroundCypress pollen is becoming an increasing cause of respiratory allergy in some regions worldwide.ObjectiveThe aim of this study was to determine some of the main allergens implicated in the common cypress (C. sempervirens) pollen allergy.MethodsPollen 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.ResultsFive 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.ConclusionThe 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.

Ability of Pollen Cytoplasmic Granules to Induce Biased Allergic Responses in a Rat Model

Background: 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. Methods: 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. Results: 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. Conclusions: 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.