Year-to-year variation of the elemental and allergenic contents of Ailanthus altissima pollen grains: an allergomic study.

The Ailanthus altissima pollen (AAP) has been reported as an emerging aeroallergen worldwide. This paper aims at examining the allergen pattern and the elemental composition of A. altissima pollen collected during two consecutive seasons (2014 and 2015). A gel-based allergomic study and SEM coupled to energy-dispersive X-ray (EDX) analysis have been carried out in order to evaluate the allergenic and elemental composition of AAP in two consecutive years. The IgE reactive patterns of 2014 and 2015 AAP PBS extracts were compared using the serum of a 31-year-old woman suffering from severe pollinosis symptoms to AAP. The EDX analysis revealed an important year-to-year variation in the ratios of some polluting elements such as nickel, sulfur, aluminum, lead, and copper. Gel alignments and comparative immunoproteomic analyses showed differential protein expression and IgE reactive patterns between AAPs collected in 2014 and 2015 pollinating seasons. From 20 distinct IgE-reactive spots detected in AAP extracts, 13 proteins showed higher expression in 2014 sample, while 7 allergen candidates exhibited an increased expression in AAP collected in 2015. Matrix-assisted laser desorption ionization-MS/MS analyses led to the identification of 13 IgE-binding proteins with confidence, all belonging to well-known allergenic protein families, i.e., enolase, calreticulin, and pectate lyase. Overall, the 2014 AAP showed higher concentrations of urban polluting elements as well as an increased expression of allergenic pectate lyase isoforms of about 52 kDa. This study demonstrates that the implementation of allergomic tools for the safety assessment of newly introduced and invasive plant species would help to the comprehensive monitoring of proteomic and transcriptomic alterations involving environmental allergens.

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.

Immunoproteomics of tree of heaven (Ailanthus atltissima) pollen allergens.

Ailanthus altissima pollen (AAP) is considered as an emerging cause of respiratory allergy in United States, Italy and Iran. However, the allergenic composition of AAP is still unknown and has yet to be characterized. The present study aimed to identify AAP allergens using a proteomics-based approach. For this purpose, optimized AAP protein extracts were analyzed using 1D- and 2D- gel electrophoresis and confronted to twenty sera from individuals with respiratory allergy during the AAP season. Candidate allergens were detected using the serum from an allergic patient with clinical history of AAP pollinosis. IgE-binding spots were identified using MALDI-TOF/TOF mass spectrometry and database searching. According to our results, AAP extracts were rich in proteins (up to 16.25mg/ml) with a molecular-weight distribution ranging from 10 to 175kDa. Two-D electrophoresis of AAP extracts revealed 125 protein spots from which 13 were IgE reactive. These IgE-binding proteins were identified as enolase, calreticulin, probable pectate lyase 6, conserved hypothetical protein and ras-related protein RHN1-like. By our knowledge, this study is the first report identifying AAP allergens. These findings will open up further avenues for the diagnosis and immunotherapy of the AAP allergy as well as for the cloning and molecular characterization of relevant allergens. BIOLOGICAL SIGNIFICANCE: Ailanthus altissima colonizes new areas every year in Iran and is spreading aggressively worldwide. According to USDA, the tree of heaven is now present as an invasive plant in 30 states in US (www.invasivespeciesinfo.gov/plants/treeheaven.shtml) and come to dominate large areas in many regions. Up to now, several cases of allergy to A. altissima pollen have been reported in United States, Italy and Iran [1-4]. However, there is still no information on the sensitizing allergens and the molecular origin of these clinical symptoms, which constitutes a serious threat to patients suffering from respiratory allergies in these regions. To our knowledge, the current study describes, therefore, the first panel of proteins responsible for IgE-mediated A. altissima pollinosis by using a gel-based proteomic approach. This work represents the pioneer proteomic investigation on Simaroubaceae spp. and provides useful insights for further studies on the allergens of this widely distributed plant family.

Kinetic of NO2 uptake by Phleum pratense pollen: chemical and allergenic implications.

Phleum pratense pollen was exposed to NO(2) in a reactor allowing a continuous analysis of NO(2) concentration by FTIR. The uptake coefficient of NO(2) on pollen was calculated postulating a first order kinetic reaction and a value of (1.1 ± 0.1) x 10(-7) was determined. NO(2) uptake was faster when the pollen water content was increased and when the pollen was pre-treated with ozone. The effect of NO(2) exposure on pollen allergic properties was investigated by quantifying Th2- and Th1-associated chemokines in a model of human dendritic cells. Cellular analysis clearly showed that cells exposed to fumigated pollen favored the production of chemokines known to promote Th2-cell responses. Altogether these data demonstrate that NO(2) uptake by pollen directly correlates with increased Th2 response in human cells,and are in favor of the involvement of NO(2) pollution in the increase of allergic diseases.

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.

Differential allergenicity of mature and immature pollen grains in Shasta daisy (Chrysanthemum maximum Ramond).

Weed pollen grains belonging to the Asteraceae family contain a variety of allergens inducing type I and IV allergies in susceptible people. The aim of this research was to compare the allergenic properties of immature and mature Shasta daisy pollen grains (Chrysanthemum maximum Ramond) to define the potential role of the maturation process on the allergenicity of Asteraceae pollen grains. The immature (IP) and mature pollen (MP) grains were first studied by optical and scanning electron microscopand their protein contents were quantitatively and qualitatively analyzed. Pollen extracts were finally used to sensitize guinea pigs in order to obtain IP and MP specific antibodies. Nasal provocation tests using IP and MP crude extracts were also performed on pre-sensitized guinea pigs. The MP extract induced IgE and eosinophilia in blood and positive skin tests in sensitized guinea pigs. Moreover, high number of eosinophils was found in the nasal mucosa of MP sensitized guinea pigs. SDS-PAGE analysis of the IP and MP protein content showed seven and five apparent bands ranging from 7 to 66kDa respectively. According to immunoblot analysis, MP extract contained a single allergen of 66kDa. The overall results showed developmental processes of Shasta daisy pollen grains towards both morphological and molecular changes increasing their allergenic potency.

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.

Comparative study of the pollen protein contents in two major varieties of Cupressus arizonica planted in Tehran.

During past few years, the Cupressus arizonica has been abundantly planted in Tehran, causing a significant increase of allergic diseases from the middle of winter to the beginning of spring. The aim of this study was the comparison of pollen protein content in two major varieties of C. arizonica planted in Tehran, including C. arizonica var. arizonica and C. arizonica var. glabra, in order to determine pollen's specificity of each variety and also to find out whether environmental conditions can influence pollen protein contents and its allergenic components. Pollen grains were directly collected from mature male cones of trees planted in different areas of the city. Pollen's proteins were extracted, and were analyzed by SDS PAGE. Total protein content of pollen extracts was measured by Bradford assay. Our investigations revealed noticeable differences in protein content of each variety. Bradford protein assay showed a higher total protein content in C. arizonica var. arizonica pollen extracts. A new major protein, with an approximate molecular weight of about 35 kDa was detected in both varieties. Immunoblotting using the serum of a cypress allergic subject showed that the protein with 35 kDa was also the major allergen of both varieties in pollen extracts. These results showed that there are some intraspecie specificities in Arizona cypress pollens. The major allergen of Cupresuss arizonica pollen, Cup a 1 (45 kDa), has been reported as the most representative protein in pollen extracts of Mediterranean countries, but in our autochthon extracts of both varieties, a protein band at 35 kDa was more representative. These observations seem to indicate that C. arizonica pollen protein content may be influenced by environmental conditions. Moreover, Immunoblot results provided a reliable indication on the allergenic activity of this new major protein band at 35 kDa. The confirmation of these aspects would facilitate the preparation of an effective extract, improving the diagnosis of the allergy to the Cupressus arizonica pollen.