Biochemical composition of Phleum pratense pollen grains: A review.

The Poaceae family is composed of 12,000 plant species. Some of these species produce highly allergenic anemophilous pollen grains (PGs). Phleum pratense pollen grains (PPPGs) emerged as a model for studies related to grass allergy. The biochemical composition of allergenic PGs has not yet been fully described despite potential health effects of PG constituents other than allergenic proteins. This review brings together the information available in literature aiming at creating a comprehensive picture of the current knowledge about the chemical composition of allergenic PGs from timothy grass. PPPGs have an average diameter between 30-35 µm and the mass of a single PG was reported between 11 and 26 ng. The pollen cytoplasm is filled with two types of pollen cytoplasmic granules (PCGs): the starch granules and the polysaccharide particles (p-particles). Starch granules have a size between 0.6-2.5 µm with an average diameter of 1.1 µm (estimated number of 1000 granules per PG) while p-particles have a size ranging around 0.3 to 0.4 µm (estimated number between 61,000-230,000 p-particles per PG). The rupture of PG induces the release of PCGs and the dispersion of allergens in the inhalable fraction of atmospheric aerosol. PPPGs are composed of sporopollenin, sugars, polysaccharides, starch, glycoproteins (including allergens), amino-acids, lipids, flavonoids (including isorhamnetin), various elements (the more abundant being Si, Mg and Ca), phenolic compounds, phytoprostanoids, carotenoids (pigments) metals and adsorbed pollutants. PPPG contains about a hundred different proteins with molecular masses ranging from 10 to 94 kDa, with isoelectric points from 3.5-10.6. Among these proteins, allergens are classified in eleven groups from 1 to 13 with allergens from groups 1 and 5 being the major contributors to Phl p pollen allergy. Major allergen Phl p 5 was quantified in PPPGs by several studies with concentration ranging from 2.7 and 3.5 µg.mg-1 in unpolluted environment. Values for other allergens are scarce in literature; only one quantitative assessment exists for allergen groups Phl p 1, 2 and 4. The extractible lipid fraction of PPPGs is estimated between 1.7-2.2% of the total PG mass. The main chemical families of lipids reported in PPPGs are: alkanes, alkenes, alcohols, saturated and unsaturated fatty acids, di- and tri-hydroxylated fatty acids, aldehydes and sterols. Several lipid compounds with potential adjuvant effects on allergy have been specifically quantified in PPPGs: E2-like prostaglandin (PGE2), B4-like leukotriene (LTB4), unsaturated fatty acids (linoleic and linolenic acids and their hydroxylated derivatives), adenosine, vitamins and phenolic compounds. Some other biochemical characteristics such as NAD(P)H oxidase, protease activity and pollen microbiome were described in the literature. The bioaccessibility in physiological conditions has not been described for most biochemicals transported by allergenic PPPGs. There is also a considerable lack of knowledge about the potential health effects of pollen constituents other than allergens. The variability of pollen composition remains also largely unknown despite its importance for plant reproduction and allergy in an environment characterized by chemical pollution, climate change and loss of biodiversity.

Update on pollen-food allergy syndrome.

INTRODUCTION: Allergies affect 20-30% of the population and respiratory allergies are mostly due to pollen grains from anemophilous plants. One to 5% of people suffer from food allergies and clinicians report increasing numbers of pollen-food allergy syndrome (PFAS), such that the symptoms have broadened from respiratory to gastrointestinal, and even to anaphylactic shock in the presence of cofactors. Thirty to 60% of food allergies are associated with pollen allergy while the percentage of pollen allergies associated to food allergy varies according to local environment and dietary habits. AREAS COVERED: Articles published in peer-reviewed journals, covered by PubMed databank, clinical data are discussed including symptoms, diagnosis, and management. A chapter emphasizes the role of six well-known allergen families involved in PFAS: PR10 proteins, profilins, lipid transfer proteins, thaumatin-like proteins, isoflavone reductases, and ß-1,3 glucanases. The relevance in PFAS of three supplementary allergen families is presented: oleosins, polygalacturonases, and gibberellin-regulated proteins. To support the discussion a few original relevant results were added. EXPERT OPINION: Both allergenic sources, pollen and food, are submitted to the same stressful environmental changes resulting in an increase of pathogenesis-related proteins in which numerous allergens are found. This might be responsible for the potential increase of PFAS.

Cypress Pollinosis: from Tree to Clinic.

Cypress (Cupressus sp.pl) is a genus within the Cupressaceae family. This family covers all of the Earth's continents except for Antarctica, and it includes about 160 species. The most important taxa for allergic diseases belong to five different genera: Cupressus, Hesperocyparis, Juniperus, Cryptomeria, and Chamaecyparis. Cupressaceae species share a common pollen type that can even include the genus Taxus (Taxaceae) when this plant is also present. As Juniperus oxycedrus pollinates in October, Cupressus sempervirens in January and February, Hesperocyparis arizonica (prev. Cupressus arizonica) in February and March, and Juniperus communis in April, the symptomatic period is long-lasting. Due to global warming, the pollination period tends to last longer, and there is a trend for Cupressaceae bioclimate niches to migrate north. In Mediterranean areas, C. sempervirens (Italian cypress or Mediterranean cypress) is by far the most common pollinating species. It accounts for half of the total pollination level. The group 1 major allergens belong to the pectate-lyase family, and members share 70 to 97% sequence homology within the different Cupressaceae. Group 2 allergens correspond to the polygalacturonase protein family, while group 3, a minor allergen, belongs to the family of "thaumatin-like proteins," a pathogenesis-related protein 5. Group 4 allergens are Ca++-binding protein (4 EF-hands). Aside from these four groups, about 15 other allergens have been reported. Prominent among these is a basic low-molecular mass cross-reactive allergen that was identified recently, and which is suspected to be involved in pollen food syndromes which are common with peach and citrus. The prevalence of cypress allergy in the general population ranges from 0.6 to 3%, depending on the degree of exposure to the pollen. Depending on the geographic area and the studied population, 9 to 65% of outpatients consulting an allergist may have sensitization to cypress pollen. Repeated cross-sectional studies performed at different time intervals have demonstrated a threefold increase in the percentage of cypress allergy around the Mediterranean area. Risk factors include a genetic predisposition and/or a strong exposure to pollen, and the natural history of cypress allergy allows identification of a subgroup of patients as allergic rather than atopic. Concerning the clinical expression, rhinitis is the most prevalent symptom, while conjunctivitis is the most disabling. Pharmacological treatment of cypress allergies is not different from that of other seasonal allergies. Immunotherapy has been used, initially by subcutaneous injections, but currently mostly through the sublingual route. Although clinical trials have included only a limited number of patients, it has proven effective and safe. Avoidance can be implemented at the individual level, as well as at the community level, through the use of alternative plants, low-pollinating cypresses, or by trimming hedges before pollination.

The Use of Combinatorial Hexapeptide Ligand Library (CPLL) in Allergomics.

The recent progress of proteomic protocols led to more efficient protein extraction and concentration procedures to remove nonprotein interfering compounds present in the starting material and to increase the concentration of underrepresented proteins. Combinatorial hexapeptide ligand libraries (CPLL) were recently applied to both plant- and animal-derived tissues for capturing the low- and very low-abundance allergens. Several IgE-binding proteins which were previously absent or poorly represented by using conventional proteomics tools have been detected and characterized through a CPLL-based approach. In the present chapter, a protocol based on improved protein extraction and enrichment by CPLL, allowing the immunochemical characterization of several "hidden allergens" in cypress pollen, is described in detail.

Urtica dioica pollen allergy: Clinical, biological, and allergomics analysis.

BACKGROUND: The most emblematic members of Urticaceae at allergic risk level are wall pellitories (Parietaria), whereas nettle (Urtica) pollen is considered as poorly allergenic. No allergen from nettle pollen has yet been characterized, whereas 4 are listed for Parietaria pollen by the International Union of Immunological Societies. Clinical and biological profiles of 2 adult men who developed symptoms against nettle pollen and/or leaves were studied. OBJECTIVE: To characterize the allergic reaction and identify the potential nettle pollen sensitizing allergens. METHODS: IgE-mediated reaction to nettle pollen extract was evaluated by skin prick test, immunoassay, nasal provocation, and basophil activation test. To characterize specific nettle pollen allergens, an allergomic (IgE immunoproteomic) analysis was performed combining 1- and 2-dimensional electrophoresis, IgE immunoblots of nettle pollen extract, identification of allergens by mass spectrometry, and database queries. RESULTS: The results of biological and immunochemical analyses revealed that the allergic rhinitis was due to Urtica dioica pollen in both patients. The allergomic analysis of nettle pollen extract allowed the characterization of 4 basic protein allergens: a thaumatin-like protein (osmotin) with a relative molecular mass of 27 to 29 kDa, a pectinesterase (relative molecular mass, 40 kDa), and 2 other basic proteins with relative molecular masses of 14 to 16 kDa and 43 kDa. There is no or only very weak allergen associations between pellitory and nettle pollen. CONCLUSION: Exposure to nettle pollen can be responsible of allergic symptoms, and several allergens were characterized. Unravelling the allergens of this underestimated allergy might help to improve diagnosis and care for patients, to predict cross-reactivities and design adapted specific immunotherapy.

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.

High IgE sensitization to maize and rice pollen in the highlands of Madagascar.

INTRODUCTION: Maize and rice are two crops constituting the main food supply in many under-developed and developing countries. Despite the large area devoted to the culture, the sensitization to the pollen from these plants is reported to be low and often considered as an occupational allergy. METHODS: Sixty five Malagasy pollen allergic patients were clinically and immunochemically investigated with regard to maize and rice pollen allergens. Pollen extracts were electrophoretically separated in 1 and 2 dimensions and IgE and IgG reactivities detected upon immunoblotting. RESULTS: When exploring the sensitization profile of Malagasy allergic patients to maize and rice pollen, it appears that a high proportion of these patients consulting during grass pollinating season were sensitized to both pollen as revealed by skin prick testing (62 vs. 59%) and IgE immunoblotting (85 vs. 40%). Several clinically relevant allergens were recognized by patients' serum IgE in maize and rice pollen extracts. CONCLUSION: The high levels of maize and rice pollen sensitization should be related, in this tropical region, to a specific environmental exposure including i) a proximity of the population to the allergenic sources and ii) a putative exacerbating effect of a highly polluted urban atmosphere on pollen allergenicity. Cross-reactivities between wild and cultivated grasses and also between rice and maize pollen are involved as well as some specific maize sensitizations. The presence of dense urban and peri-urban agriculture, in various African regions and worldwide, could be a high environmental risk factor for people sensitive to maize pollen.

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.

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.

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.

Proteomic analysis of major and minor allergens from isolated pollen cytoplasmic granules.

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.

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.

A modified enzyme-linked immunosorbent assay adapted for immunodetection of low amounts of water-insoluble proteins.

A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l(-1) thiourea, 7 mol l(-1) urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60-500 ng ml(-1)) of water-insoluble proteins extracted with high concentrations of TUC.