Anaphylaxis to Vespa velutina nigrithorax: Pattern of Sensitization for an Emerging Problem in Western Countries.

OBJECTIVE: To define the sensitization pattern of patients with anaphylaxis to Vespa velutina nigrithorax (VVN). METHODS: We studied 100 consecutive Spanish patients with anaphylaxis to Hymenoptera venom and systematically determined specific IgE (sIgE) to whole venoms (Vespula species, Polistes dominula, Apis mellifera, Vespa crabro, and Dolichovespula maculata) and their molecular components (rApi m 1, rApi m 5, rApi m 10, rVes v 1, rVes v 5, rPol d 5, and cross-reactive carbohydrates). Specific IgE to VVN venom and its antigen 5 (nVesp v 5) were measured in a subsample. RESULTS: Seventy-seven patients had anaphylaxis to VVN. Of these, only 16 (20.8%) reported previous VVN stings, but were stung by other Hymenoptera. Positive sIgE (>0.35 kUA/L) to each of the whole venoms was detected in >70% of patients (Vespula species in 100%). The components showing >50% positivity were rApi m 5 (51.4%), rPol d 5 (80.0%), and rVes v 5 (98.7%). This pattern was similar to that of Vespula species anaphylaxis (n=11) but different from that of A mellifera anaphylaxis (n=10). Specific IgE to nVesp v 5 was positive in all patients (n=15) with VVN anaphylaxis and was correlated with sIgE to both rVes v 5 (R=0.931) and rPol d 5 (R=0.887). CONCLUSIONS: VVN has become the commonest cause of Hymenoptera anaphylaxis in our area. Most cases report no previous VVN stings. Their sensitization pattern is similar to that of patients with anaphylaxis to other Vespidae. Specific IgE to antigen-5 from VVN, Vespula species, and P dominula are strongly correlated in patients with VVN anaphylaxis.

Component-resolved diagnosis of vespid venom-allergic individuals: phospholipases and antigen 5s are necessary to identify Vespula or Polistes sensitization.

BACKGROUND: Cross-reactivity between hymenoptera species varies according to the different allergenic components of the venom. The true source of sensitization must therefore be established to ensure the efficacy of venom immunotherapy. OBJECTIVE: In the Mediterranean region, Polistes dominulus and Vespula spp. are clinically relevant cohabitating wasps. A panel of major vespid venom allergens was used to investigate whether serum-specific IgE (sIgE) could be used to distinguish sensitization to either vespid. METHODS: Fifty-nine individuals with allergic reactions to vespid stings and positive ImmunoCAP and/or intradermal tests to vespid venoms were studied. sIgE against recombinant and natural venom components from each wasp species was determined using the ADVIA Centaur(®) system. RESULTS: sIgE against recombinant antigen 5s sensitization to be detected in 52% of the patients tested (13/25). The sensitivity increased to 80% (20/25), when using natural antigen 5s, and to 100% with the complete panel of purified natural components, because the sIgE was positive to either the antigen 5s (Pol d 5/Ves v 5) or to the phospholipases (Pol d 1/Ves v 1) of the two vespids, or to both components at the same time. In 69% of cases, it was possible to define the most probable sensitizing insect, and in the rest, possible double sensitization could not be excluded. Vespula hyaluronidase was shown to have no additional value as regards the specificity of the assay. CONCLUSIONS: The major allergens of P. dominulus' and Vespula vulgaris' venom, namely phoshpholipases and antigen 5s, are required to discriminate the probable sensitizing species in vespid-allergic patients.

Assessment of airborne soy-hull allergen (Gly m 1) in the Port of Ancona, Italy.

BACKGROUND: Epidemic asthma outbreaks are potentially a very high-risk medical situation in seaport towns where large volumes of soybean are loaded and unloaded Airborne allergen assessment plays a pivotal role in evaluating the resulting environmental pollution. OBJECTIVE: The aim of this study was to measure the airborne Gly m 1 allergen level in the seaport of Ancona in order assess the soybean-specific allergenic risk for the city. METHODS: Allergen and PM10 were evaluated at progressive distances from the port area. Allergen analysis was performed by monoclonal antibody-based immunoassay on the sampled filters. Daily meteorological data were obtained from the local meteorological station. For estimating the assimilative capacity of the atmosphere, an approach based on dispersive ventilation coefficient was tried. RESULTS: The allergen concentrations detected were low (range = 0.4-171 ng/m3). A decreasing gradient of the airborne allergen from the unloading area (22.1 +/- 41.2 ng/m3) to the control area (0.6 +/- 0.7 ng/m3) was detected. The concentration of the airborne Gly m 1 was not coupled with the presence of the soy-carrying ships in the port. A statistically significant relationship between airborne allergen, PM10 and local meteorological parameters quantifies the association with the atmospheric condition. CONCLUSION: Airborne Gly m 1 is part of the atmospheric dust of Ancona. The low level of this allergen seems consistent with the absence of asthma epidemic outbreak.

Profilin sensitization detected in the office by skin prick test: a study of prevalence and clinical relevance of profilin as a plant food allergen.

BACKGROUND: Profilin, a pan-allergen present in all eukaryotic cells, is one of the main causes of cross-sensitization between pollen and plant-derived foods, but its clinical relevance as a food allergen is still debated. OBJECTIVE: To investigate the prevalence of profilin sensitization in a pollen-allergic population and its clinical relevance as a food allergen. METHODS: Two hundred consecutive patients with pollen allergy underwent skin prick tests (SPT) with purified natural date palm profilin (Pho d 2; 50 microg/mL; Alk Abello, Madrid, Spain). Those reporting adverse reactions to foods (confirmed by SPT with either commercial food extracts or fresh foods) underwent SPT with an apple extract containing uniquely Mal d 1 (2 microg/mL; ALK-Abello), and with a commercial peach extract containing uniquely lipid transfer protein (LTP 30 microg/mL; ALK-Abello). RESULTS: Sixty patients (30%) showed skin reactivity to date palm profilin, Pho d 2. All were sensitized to grass pollen, and most of them reacted to birch, mugwort, ragweed and plantain pollen as well. SPT with pellitory and cypress scored negative in a high proportion of profilin reactors [26/60 (43%) and 33/60 (55%), respectively]. More than one half (34/60 [57%]) of profilin reactors had food allergy; 21 of these were monosensitized to profilin, 11 were sensitized to both profilin and Bet v 1 homologous protein, one to both profilin and LTP, and one to all the three allergens. The large majority of profilin-allergic patients reported oral allergy syndrome as the only food-induced symptom and were able to tolerate the offending foods if they were cooked or otherwise processed. Twenty-eight of 34 reported reactivity to two or more plant-derived foods. Rosaceae, tree nuts, melon and watermelon, tomato, pineapple, citrus fruits and banana were the more frequently offending foods. CONCLUSION: Profilin should be considered a clinically relevant food allergen. Allergy to melon, watermelon, tomato, banana, pineapple and orange may be considered as a marker of profilin hypersensitivity. This study underlines the clinical importance of being able to diagnose hypersensitivity to single food allergenic proteins by SPT, particularly when the relevant food allergen sources contain several allergens that show different chemical/physical features and, hence, completely different risk profiles.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Structural analysis of the small chain of the 2S albumin, napin nIII, from rapeseed. Chemical and spectroscopic evidence of an intramolecular bond formation.

Napin nIII is a 2S albumin from rapeseed (Brassica napus L.) homologous to the major mustard allergen. It is composed of two different polypeptide chains linked by two disulphide bonds. The small chain has been isolated by reverse-phase HPLC after reduction of the native protein and its primary structure elucidated. This 37 residue polypeptide contains only two cysteines, at positions 10 and 23, which show a great tendency to form a non-native intramolecular disulphide bridge. The kinetic analysis of this process was performed by measuring the fluorescence emission of the single tryptophan residue of the molecule since its fluorescence intensity is about 30% decreased during disulphide formation. Small changes on the secondary structure of the polypeptide were measured by circular dichroism. The process is delayed in the presence of the reduced large chain of nIII. However, no dimer formation was detected under the conditions used, either between small chains or between the small and the large chains. Thus, the interchain disulphide formation in napin nIII should be considered as an early step during maturation of this multi-subunit seed protein.

Beta-turns as structural motifs for the proteolytic processing of seed proteins.

Fifteen NH2- and COOH-terminal ends from both small and large chains of the most abundant 2 S albumins from Brassica napus seeds have been sequenced. This allows the determination of the exact proteolytic maturation sites of these proteins. Each one of these proteins arises from a polypeptide precursor which is cleaved during the post-translational processing at four sites, giving two different chains linked by disulphide bridges on the mature 2 S albumin. The hydrolyzed bonds involved in the processing are located in proline and glycine-rich regions, forming tetra-peptides with a very high beta-turn probability. Similar results have been found through the analysis of the 2 S albumin precursors from other seeds. These facts are interpreted in terms of the existence of a beta-turn specific endoprotease activity involved in the maturation process of 2 S albumins.

A new distinct group of 2 S albumins from rapeseed. Amino acid sequence of two low molecular weight napins.

Two napins (nIa and nIb), isolated from Brassica napus (rapeseed) seeds, have been sequenced. The two proteins show the common structural pattern of the 2 S albumins, since they are composed of two disulfide-linked chains of different size, yet they exhibit an atypical low molecular weight (12.5 kDa vs. 14.5 kDa of the major napins). High sequence similarity has been found between these 2 proteins, but only 54% similarity can be estimated from their comparison with the 14.5 kDa major napins. Thus, nIa and nIb are considered representatives of a new distinct group of rapeseed napins since all the previously known napins exhibit 95% sequence similarity. Unexpectedly, the similarity increases when compared with the 2 S proteins from other species.

Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast.

Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast alpha signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis).

Expression of yellow jacket and wasp venom Ag5 allergens in bacteria and in yeast.

Antigen 5 (Ag5), of unknown biological function, is one of the major venom allergens of vespids and fire ants. We have compared the expression of Ag5 in bacteria and in yeast. Recombinant Ag5 from bacteria formed an insoluble intracellular product, which was not properly folded, but that produced in Pichia pastoris was secreted to the extracellular medium. Immunochemical characterizations showed the secreted Ag5 to have the native structure of the natural protein. This is of interest since the B cell epitopes of Ag5 are mainly of the discontinuous type. These studies were made with Ag5s from yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis), and with hybrid Ag5 molecules that contained partial sequences of these two species. In vitro allergenicity studies with sera from yellow jacket-sensitive patients showed that some of these hybrid molecules had a greatly reduced allergenicity but retained the immunogenicity of the natural allergen. This could be of importance for immunotherapy of this type of allergy.

Cross-reactivity between the major allergen from olive pollen and unrelated glycoproteins: evidence of an epitope in the glycan moiety of the allergen.

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies.

The spectrum of olive pollen allergens.

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 - Ole e 1 to Ole e 8 - have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca(2+)-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.

Isolation and characterization of a major allergen from oriental mustard seeds, BrajI.

A 2 S albumin from oriental mustard (Brassica juncea) seeds has been isolated and characterized as an allergen. This protein, BrajI, was recognized by human IgE from mustard-sensitive individuals, as proved by using two different enzyme immunoassays. BrajI was found to be closely related to Sin a I, the major allergen from yellow mustard seeds. Many fractions with molecular weights ranging from 16,000 to 16,400 and with differences in charge were separated by ion-exchange chromatography. They exhibited small but significant amino acid composition differences for Glx, Val, Ile, Lys, and Arg contents. The heterogeneity of BrajI can be explained by size and charge differences of its heavy and light chains. All of the isoallergenic forms of BrajI gave a single precipitation band in double diffusion immunoassays when using a Sin a-I-specific rabbit polyclonal serum.

1H NMR assignment and global fold of napin BnIb, a representative 2S albumin seed protein.

Napin BnIb is a representative member of the 2S albumin seed proteins, which consists of two polypeptide chains of 3.8 and 8.4 kDa linked by two disulfide bridges. In this work, a complete assignment of the 1H spectra of napin BnIb has been carried out by two-dimensional NMR sequence-specific methods and its secondary structure determined on the basis of spectral data. A calculation of the tertiary structure has been performed using approximately 500 distance constraints derived from unambiguously assigned NOE cross-correlations and distance geometry methods. The resulting global fold consists of five helices and a C-terminal loop arranged in a right-handed spiral. The folded protein is stabilized by two interchain disulfide bridges and two additional ones between cysteine residues in the large chain. The structure of napin BnIb represents a third example of a new and distinctive folding pattern first described for the hydrophobic protein from soybean and nonspecific lipid transfer proteins from wheat and maize. The presence of an internal cavity is not at all evident, which rules out in principle the napin BnIb as a carrier of lipids. The determined structure is compatible with activities attributed to these proteins such as phospholipid vesicle interaction, allergenicity, and calmodulin antagonism. Given the sequence homology of BnIb with other napins and napin-type 2S albumin seed proteins from different species, it is likely that all these proteins share a common architecture. The determined structure will be crucial to establish structure-function relationships and to explore the mechanisms of folding, processing, and deposition of these proteins. It will also provide a firm basis for a rational use of genetic engineering in order to develop improved transgenic plants.

Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

Solution structure of allergenic 2 S albumins.

The NMR solution structures at different levels of refinement of three different 2 S albumin seed proteins, the recombinant pronapin precursor from Brassica napus, the recombinant RicC3 from Ricinus communis and the methionine-rich protein from sunflower ( Helianthus annuus ), are described. The resulting common structure consists of a bundle of five alpha-helices, folded in a right-handed superhelix. The structure is very similar to that of other plant proteins: the hydrophobic protein from soybean, non-specific lipid transfer proteins and amylase/trypsin inhibitors. Analogies and differences in the structures of these families, as well as their possible relationship to allergenicity, are discussed.

Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity.

Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.

IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1.

BACKGROUND: Pollen from olive trees (Olea europaea ) is a cause of pollinosis and an aggravating of asthma in Mediterranean regions. Recently, Ole e 1, the major allergen from olive tree pollen, has been isolated and its amino acid sequence has been elucidated. It is a glycoprotein whose carbohydrate moiety is involved in an IgE-binding epitope responsible for cross-reactivity among plant glycoproteins. However, the allergenicity of the free carbohydrate side chains remains to be clarified. OBJECTIVE: The purpose of this study was to isolate the main carbohydrate component of Ole e 1 allergen and analyze its IgE-binding and histamine-release capabilities. METHODS: Deglycosylation treatment of Ole e 1 with PNGase F and gel exclusion chromatography were used to isolate the main sugar component of the allergen. Sera of patients who are allergic to olive pollen and sera sensitive to Ole e 1 have been used in dot blotting assays of IgE binding to the isolated carbohydrate. Heparinized whole blood obtained from patients sensitive to Ole e 1 were stimulated by the free carbohydrate; the resulting histamine release was measured. RESULTS: The main sugar component of Ole e 1 has been isolated. Free carbohydrate was able to bind IgE from sera of patients allergic to olive pollen; the sera of 65% of these patients contained anticarbohydrate reacting IgE, and 100% of those patients were sensitive to Ole e 1. The free carbohydrate promoted in vitro histamine release from basophils of sensitized patients. CONCLUSION: The carbohydrate moieties of allergenic glycoproteins can constitute significant determinants on the binding to IgE of the sera from patients who are hypersensitive and can be responsible for inducing histamine release from blood cells.