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Background Grasses and olive trees are the most common sources of allergenic pollen worldwide. Although they share some allergens, there are few studies analyzing the in vitro cross-reactivity between them. The aim was to define the cross-reactivity between Olea europaea and Phleum pratense using well-characterized sera of allergic children from Madrid, Spain.Methods 66 patients (mean age 10.32+/−4.07 years) were included in the study. All suffered from rhinoconjuntivitis and/or asthma and had a positive skin test and/or specific IgE determination to olive and grass pollen. Serum sIgE to individual allergens was conducted and sIgE against different grass species and olive was also determined by ELISA. Inhibition assays were performed using two serum sources, containing, or not, sIgE to minor allergens. Mass spectrometry analysis was performed in both extracts.Results 59/66 (89.39%) children had a positive sIgE determination by ELISA to grasses and 57/66 (86.36%) to olive pollen. There was no significant correlation between sIgE levels to grass and olive. Inhibition assays demonstrated no cross-reactivity between P. pratense and olive pollen when using the pool containing mainly sIgE to major allergens, whereas minimal to moderate cross-reactivity was detected when the serum contained high sIgE titers to minor allergens. Proteomic analyses revealed the presence of 42 common proteins in grasses and olive pollens.Conclusion No in vitro cross-reactivity was observed when sIgE was mainly directed to major allergens. In our population, sensitization to olive and grasses is not due to cross-reactivity. The contribution of the major allergens seems to be determinant. Keywords: Cross-reactivity, Minor allergens, Olea europaea, Panallergen, Phleum pratense, Pollen

Background Allergoids were first used in the decades of the 60s and 70s of the last century as an effective treatment of allergic respiratory diseases. Allergoids can be modified with formaldehyde or glutaraldehyde. Modified allergens, or allergoids, decrease the risk of adverse reactions while administering higher allergen doses. The objective of this study was to analyse specific IgE and IgG binding to glutaraldehyde modified and non-modified allergen extracts of Phleum pratense. Methods The sera of 69 patients sensitized to P. pratense were tested. All these patients had signs and symptoms of rhinoconjunctivitis with, or without, asthma in May and June of 2011. All these patients had positive skin prick tests to a standardized extract of P. pratense, and other grass species. Most patients were also sensitized to olive pollen. Specific IgE and IgG binding were analysed by direct ELISA against P. pratense native (non-modified) and allergoid extracts. Relative potencies were evaluated through ELISA inhibition assays, and the protein composition of non-modified and allergoid samples was determined by Mass Spectrometry (MS/MS). Results Mean Specific IgE levels against the native extract was 16.68 ± 11.65 Units (U) and against the allergoid: 7.26 ± 8.24 U (P < 0.0001; Mann-Whitney). On the other hand, mean specific IgG binding against the non-modified extract was 90.34 ± 75.57 U versus 76.19 ± 70.31 U against the allergoid (P = 0.16; Mann-Whitney). Linear regression coefficients obtained between immunoglobulin reactivity against both extracts were: r2 = 0.51 for specific IgE and r2 = 0.83 for specific IgG. An important decrease in the allergenic activity, measured by inhibition ELISA, was clearly observed. The MS/MS assay revealed the presence of the mayor allergen, and some isoforms, in non-modified and allergoid extracts. Conclusions Results obtained demonstrate that the glutaraldehyde polymerization process induces an important decrease in specific IgE binding to allergoids of P. pratense while there are no significant differences in specific IgG binding. The allergenic composition of the P. pratense allergoid was equivalent to the non-modified pollen extract.

Background The most common allergenic pollens in patients with pollinosis in Central Spain are grasses and olive pollen, with a prevalence of positive skin prick test results of 94 and 61%, respectively. Shared proteins have been described in olive and grasses, such as profilins, polcalcin and trypsin inhibitors, but there are few in vitro studies analyzing the potential cross reactivity between both species. The aim of the present study was to analyze the protein composition of both allergen extracts, and the allergenic cross-reactivity between O. europaea and different grass pollen species. Methods Seventy-two (72) patients (mean age 10.4 years) were included in the study. All of them suffered rhinoconjuntivitis and/or asthma and were sensitized to olive and/or grass pollen. Specific IgE of the individual patients against O. europaea, and to the grass species: Dactylis glomerata, Phleum pratense and Trisetum paniceum were determined by ELISA. Inhibition assays were performed to verify allergenic crossreactivity between grass species and olive. Mass spectrometry analysis was performed to characterize the extracts and establish if there are common proteins in both, grass and olive pollens, that could act as cross reactive proteins. Results Three different sensitization patterns were observed: 1) sensitization to olive and grass pollen, 2) sensitization to olive and not to grasses and 3) sensitization to grasses and not to olive. Different pools of sera were mixed according to this classification and used for the different assays. Correlation coefficients found for the 3 grass species were significant (P < 0.0001; Spearman), but not for olive pollen (P = 0.14; Spearman). Proteomic analysis revealed the presence of more than 40 common proteins in grasses and olive pollens, but inhibition assays demonstrated no allergenic cross-reactivity between both families. Conclusions There is no in vitro crossreactivity between O. europaea and Grass pollen extracts, in spite of the allergens and the large number of common proteins shared by these pollens. We can conclude that sensitization to olive and grasses is species specific.

Background Sensitization to Betula verrucosa pollen is common in North Central Europe. In Spain, it is also common in patients from Galicia, a region located in the Northwest of Spain. Birch trees are abundant in this region and several birch species have been described. The main objectives were to determine the differences in the IgE reactivity to B. verrucosa and rBet v 1 between patients from Sweden and Spain, as well as the contribution of the major allergen Bet v 1 to the overall reactivity of B. verrucosa. Allergenicity was also compared using a serum pool derived from sera of North American (USA) patients. Methods IgE reactivity to B. verrucosa and rBet v 1 was measured in sera from 44 Spanish and 21 Swedish patients. rBet v 1 was produced as N-terminal His-tagged fusion protein and purified as originally described. All of them were sensitized to the pollen of this tree species and suffered from allergic rhinitis during the pollination period. rBet v 1 contribution was determined by inhibition ELISA using 3 pools of sera: from Spanish, North American and Swedish patients. IgE binding pattern was evaluated by Western blots using the same pools of sera. Specific IgE binding was expressed in arbitrary units. Results Specific IgE to B. verrucosa was detected in 50 from the 65 sera analyzed (mean = 3.77 ± 3.71 Units). Specific IgE levels to rBet v 1 were 3.13 ± 3.32 Units. Immunoblot assays confirmed specific IgE binding to rBet v 1, and also to other allergens present in the extract. The Spanish pool presented reactivity to more allergens than the Swedish pool. ELISA Inhibition assays, performed with a native extract and rBet v 1, revealed a significant contribution (>80% inhibition) of rBet v 1 to the allergenicity of the extract, with no differences according to the origin of the sera. Conclusions Bet v 1 has a great importance in birch allergy in Galicia and Sweden. Nevertheless, there are differences in the IgE recognition pattern according to the rest of birch allergens. Mayor allergen from birch, Bet v 1, significantly contributes to the allergenicity of B. verrucosa in Galicia.

Background D. pteronyssinus and B. tropicalis are the most common house dust mite species worldwide. D. pteronyssinus and B. tropicalis are the most prevalent species in the tropics. Twelve allergens have been described in B. tropicalis and fifteen in D. pteronyssinus. The objectives of this study were: 1) to analyze the prevalence of sensitization to B. tropicalis and D. pteronyssinus on the tropical Island of Martinique; 2) to evaluate the allergenic crossreactivity between D. pteronyssinus and B. tropicalis and 3) to identify common, and species specific allergens, using mass spectrometry (MS). The serum of patients sensitized to a single, or to both species, was further analyzed by western blots. Methods The sera of 1,243 consecutive patients evaluated for allergic rhinitis and/or asthma were evaluated for specific IgE using direct ELISA. All patients resided on the tropical island of Martinique. Crossreactivity between both species was analyzed by ELISA Inhibitions. Proteins of both species were digested with trypsin and analyzed by MS/MS (MALDI TOF/TOF). Different databases were used for the identification of the proteins. Results Sensitization to B. tropicalis was detected in 868 sera and to D. pteronyssinus in 919 sera; 135 were sensitized exclusively to B. tropicalis and 177 to D. pteronyssinus. Specific IgE levels to D. pteronyssinus were significantly higher than to B. tropicalis (Mean 0.67 OD ± 0.89 vs. 0.86 ± 1.04; P < 0.001). The correlation coefficient between specific IgE levels to both species was 0.135. ELISA Inhibition studies demonstrated minimal crossreactivity between both species. Western blots done with the serum of poly or monosensitized patients revealed specific IgE binding to similar proteins. Proteomic analysis revealed the presence of the following allergens: B. tropicalis: groups 2, 3, 4, 6, 9, 14 and groups 1, 2, 3, 4, 8, 11, 14, 15, 16 and 18 in D. pteronyssinus. Species specific, as well as common proteins were detected. Conclusions There is limited crossreactivity between B. tropicalis and D. pteronyssinus. Sensitization to their allergens seems to be a parallel phenomenon. Allergen extracts of both species are needed for the correct diagnosis and treatment of mite allergic individuals in the Caribbean.

Background Allergoids have been successfully used in the treatment of respiratory allergic diseases. They are modified allergen extracts that allow the administration of high allergen doses, due to their reduced IgE binding capacity.They maintain allergen-specific T-cell recognition. Since they are native allergen extracts that have been polymerized with glutaraldehyde, identification of the allergenic molecules requires more complicated methods. The aim of the study was to determine the qualitative composition of different polymerized extracts and investigate the presence of defined allergenic molecules using Mass spectrometry. Methods Proteomic analysis was carried out at the Proteomics Facility of the Hospital Nacional de Parapléjicos (Toledo, Spain). After reduction and alkylation, proteins were digested with trypsin and the resulting peptides were cleaned using C18 SpinTips Sample Prep Kit; peptides were separated on an Ultimate nano-LC system using a Monolithic C18 column in combination with a precolumn for salt removal. Fractionation of the peptides was performed with a Probot microfraction collector and MS and MS/MS analysis of offline spotted peptide samples were performed using the Applied Biosystems 4800 plus MALDI TOF/TOF Analyzer mass spectrometer. ProteinPilot Software V 2.0.1 and the Paragon algorithm were used for the identification of the proteins. Each MS/MS spectrum was searched against the SwissProt 2010_10 database, Uniprot-Viridiplantae database and Uniprot_Betula database. Results Analysis of the peptides revealed the presence of native allergens in the polymerized extracts: Der p 1, Der p 2, Der p 3, Der p 8 and Der p 11 in D. pteronyssinus; Bet v 2, Bet v 6, Bet v 7 and several Bet v 1 isoforms in B. verrucosa and Phl p 1, Phl p 3, Phl p 5, Phl p 11 and Phl p 12 in P. pratense allergoids. In all cases, potential allergenic proteins were also identified, including ubiquitin, actin, Eenolase, fructose-bisphosphate aldolase, luminal-binding protein (Heat shock protein 70), calmodulin, among others. Conclusions The characterization of the allergenic composition of allergoids is possible using MS/MS analysis. The analysis confirms the presence of native allergens in the allergoids. Mayor allergens are preserved during polymerization.