Optimization of the diagnosis and characterization of gibberellin-regulated protein sensitization: An Italian cohort study.

BACKGROUND: Pru p 7 was the first gibberellin-regulated protein (GRP) to be identified as a food allergen as the basis of a pollen food allergy syndrome. OBJECTIVE: To clinically and biologically characterize a group of patients with suspected allergy to Pru p 7 to optimize the diagnostic workup of GRP sensitization. METHODS: Allergy to Pru p 7 was suspected in the presence of a systemic allergic reaction to plant food, positive skin prick test results for cypress pollen and lipid-transfer protein-enriched peach extract, and absence of Pru p 3-specific immunoglobulin E. Controls were patients with food allergies, patients sensitized to Pru p 3, and patients with cypress allergy without food allergy. Diagnostic workup included skin tests, basophil activation test, Western blot, and single and multiplex assays. RESULTS: In total, 23 patients and 14 controls were enrolled. The most implicated food was peach (91.3%). Approximately 70% of patients reacted to multiple foods. Mueller 4 reactions were 8.7%. In 26.1% of cases, a cofactor triggered the reaction. The basophil activation test results were positive for rPru p 7 in 87% of the patients. Specific immunoglobulin E to Pru p 7 was detected in 95.7% by singleplex and in 73.9% by multiplex assays in patients with suspected allergies; 73.9% of them also reacted to cypress pollen GRP (Cup s 7) in Western blot analysis. CONCLUSION: Patients with Pru p 7-Cup s 7 allergy in our cohort confirm a mild-to-severe clinical syndrome characterized by pollen and food allergy. The diagnosis may benefit from the proposed selection criteria that can be used as preliminary steps to further characterize the cross-reactive GRP sensitization.

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.

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.

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.

Capsicum Allergy: Involvement of Cap a 7, a New Clinically Relevant Gibberellin-Regulated Protein Cross-Reactive With Cry j 7, the Gibberellin-Regulated Protein From Japanese Cedar Pollen.

The Capsicum genus belongs to the Solanaceae family. Bell or chili peppers are consumed worldwide, but allergy to Capsicum is rare. It is involved in the celery-birch-mugwort-spice syndrome and cross-reactivities were reported with latex. Several allergens have been described, but only 2 are referenced in the World Health Organization/International Union of Immunological Societies allergen data bank, a thaumatin-like protein and a profilin. A patient allergic to bell/chili pepper, peach, orange and Japanese cedar pollen was clinically and biologically analyzed including direct and competitive immunoblots and basophil activation tests (BATs) with allergenic source extracts and recombinant gibberellin-regulated proteins (GRPs). The patient was shown to be sensitized to Cap a 7, the GRP of Capsicum annuum newly described herein. Cross-reactivities were demonstrated between various GRPs from bell/chili pepper, peach, orange and Japanese cedar pollen either in native form in the different extracts or as recombinant allergens. A similar immunoglobulin E reactivity was found also in Capsicum chinense and against snakin-1, the GRP from potato. The patient showed a positive BAT with recombinant Cry j 7, Pru p 7 and Cap a 7, but not with recombinant snakin-1. Despite the ubiquitous nature of GRPs in plants and the immunochemical cross-reactivity observed between different GRPs, clinically relevant sensitization to this protein family seems restricted to some allergenic sources, often associated with Cupressaceae pollen allergy, and to some patients, therefore reflecting very specific and peculiar mechanisms of conditional sensitization.

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.

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Two Different Composite Markers Predict Severity and Threshold Dose in Peanut Allergy.

BACKGROUND: Safe and cost-effective biological surrogate markers to evaluate the severity and threshold dose of peanut allergy (PA) reactions during an oral food challenge (OFC) are lacking. OBJECTIVE: To evaluate biological markers associated with the severity and threshold dose of an allergic reaction during an OFC in a population of children with PA. METHODS: Demographic and biological parameters of children with peanut OFC and basophil activation test (BAT) results were collected. Patients were stratified into 2 severity groups (mild-to-moderate and severe) and 2 cumulative threshold dose groups: low (LCTG) ≤100 mg crushed peanut and high >100 mg. RESULTS: Among the 68 children included, there was a 96% concordance between the OFC and BAT result for the diagnosis of PA. Of the 56 children with a positive OFC and BAT to peanut (median age: 8.8 years), the severity of an allergic reaction and the cumulative threshold dose were not correlated (P = .24). Higher Ara h 2-specific IgE and FcεRI-positive control values were both associated with severe reactions to peanut. Combining these 2 markers led to a 92% sensitivity (84%-97%) and an 82% specificity (71%-89%) for severe reactions in all subjects. For children in the LCTG, a 4-variable composite marker, including age, normalized basophil sensitivity (EC50), and FcεRI- and fMLP-positive control values, resulted in a 97% sensitivity (89%-99%) and 61% specificity (49%-71%). CONCLUSION: Distinct composite markers including BAT allergen-specific and non-allergen-specific parameters appear to be associated with severity and cumulative threshold dose in children with PA.

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.

Monodisperse colloids synthesized with nanofluidic technology.

Limitations in the methods employed to generate micrometric colloidal droplets hinder the emergence of key applications in the fields of material science and drug delivery. Through the use of dedicated nanofluidic devices and by taking advantage of an original physical effect called capillary focusing, we could circumvent some of these limitations. The nanofluidic (i.e., submicrometric) devices introduced herein are made of soft materials, and their fabrication relies upon rapid technologies. The objects that we have generated are simple droplets, multiple droplets, particles, and Janus particles whose sizes lie between 900 nm and 3 microm (i.e., within the colloidal range). Colloidal droplets have been assembled on-chip into clusters and crystals, yielding discrete diffraction patterns. We illustrate potential applications in the field of drug delivery by demonstrating the ability of multiple droplets to be phagocytosed by murine macrophage-type cells.

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.

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation.

BACKGROUND: Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. OBJECTIVES: This work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. METHODS: A dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors (<24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 °C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. RESULTS: This streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low intra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 5 µL of blood per point could be used. CONCLUSIONS: A whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required.

Double One-Dimensional Electrophoresis (D1-DE) Adapted for Immunoproteomics.

The classical proteomics approach for the identification of allergen candidates consists on the separation of proteins by high-resolution two-dimensional electrophoresis (2-DE) with subsequent IgE immunoblotting and further analysis of IgE-reactive protein spots with mass spectrometry. In this approach at least two gels most be run. One gel is used for staining and the other is for immunoblotting by antibodies labeled with specific immunostains. Additional functional characterizations require either protein purification or 2-DE replicates and appear to be time- and reagent-consuming. Here we described a modified double one-dimensional electrophoresis (D1-DE) allowing the conversion of a protein spot previously visualized by 2-DE into an extended protein band. In D1-DE, the purity of the protein of interest is similar to 2-DE spots, but its abundance is many times higher than what can be found in a 2-DE single spot allowing many other functional analyses from a single D1-DE separation.

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.

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.