Search

Background and Objectives: Background: Cypress and olive pollen are the most prevalent sensitizers in the Mediterranean area. Some patients exhibit dual sensitization, which has not been well documented to date. Objective: To identify the allergens involved in dual cypress and olive allergy (C+O) and to study the relationship between phenotype and allergen sensitization., Methods: C+O patients were selected. Those monosensitized to olive or cypress were used as a reference. Specific IgE to whole extracts and purified allergens from olive and cypress was determined. Immunoblotting was performed to analyze IgG and IgE binding using olive polyclonal antibodies and patients' sera, respectively. Mutual immunoblotting inhibition of olive and cypress extracts and inhibition of cypress extract immunoblotting with olive allergens were performed. Multiple correspondence analysis and hierarchical cluster classifications were conducted to analyze the relationships between the clinical presentation of C+O (symptoms, seasonality) and allergen profile., Results: C+O patients were clustered in 4 phenotypes. The most frequent one (58.4%) was rhinoconjunctivitis in winter (February) and spring (May), with asthma in 38% of patients. Ole e 1 and Cup s 1 were the major allergens. Proteins homologous to Ole e 1, Ole e 9, and Ole e 11 in cypress pollen were identified and shown to inhibit IgE binding to cypress extract., Conclusions: The exclusive C+O results from cosensitization to Cup s 1 and Ole e 1 and cross-reactivity due to previously unreported Ole e 1-like, Ole e 9-like, and Ole e 11-like allergens in cypress pollen. Our findings point to 4 clinical phenotypes of winter and/or spring or perennial rhinoconjunctivitis with and without asthma.

Immunoglobulin E (IgE)-mediated allergy, which affects more than 30% of the population, is the most prevalent hypersensitivity illness. In an atopic individual, even a small amount of allergen exposure can cause IgE antibodies to be produced. Due to the engagement of receptors that are highly selective for IgE, even tiny amounts of allergens can induce massive inflammation. This study focuses on the exploration and characterization of the allergen potential of Olea europaea allergen (Ole e 9) affecting the population in Saudi Arabia. A systematic computational approach was performed to identify potential epitopes of allergens and complementary determining regions of IgE. In support, physiochemical characterization and secondary structure analysis unravel the structural conformations of allergens and active sites. Epitope prediction uses a pool of computational algorithms to identify plausible epitopes. Furthermore, the vaccine construct was assessed for its binding efficiency using molecular docking and molecular dynamics simulation studies, which led to strong and stable interactions. This is because IgE is known to play a role in allergic responses, which facilitate host cell activation for an immune response. Overall, the immunoinformatics analysis advocates that the proposed vaccine candidate is safe and immunogenic and therefore can be pushed as a lead for in vitro and in vivo investigations.Communicated by Ramaswamy H. Sarma.

Background: The so-called "thunderstorm asthma" (TA) is an uncommon but dramatic outbreak of asthma attacks occurring during a thunderstorm in the pollen and moulds season. Mechanisms which make the pollen able to enter the deeper airways and provoke severe asthma symptoms are still unclear., Objective: To test the hypothesis that sub-pollen particles (SPPs) originated from the rupture by an osmotic shock of pollen associated with TA contain allergens., Methods: After hydration, SPPs released from pollen grains of grass, pellitory, olive, cypress, ragweed and birch were isolated and determined by microscopy. Allergens were determined by in vitro ELISA inhibition tests indirectly using the sera from 10 polyreactive patients. An inhibition <50% was considered as negative, 50%-75% moderate and > 75% complete., Results: The inhibition experiments showed that the SPPs from birch and cypress were unable to inhibit serum IgE reactivity to Bet v 1 and Cup a 1, respectively. Ragweed SPPs inhibited ragweed pollen extract and Amb a 1 by 75.8 ± 0.11% and 81.2 ± 0.15%, respectively. Olive and pellitory SPPs retained almost the whole IgE-binding capability in all cases tested. Grass SPPs inhibited 32 ± 0.06% of Lolium perenne Lol p 1 and 65% of Phleum pratense extracts, but results were highly variable for individual allergens (97.5%-0.03% for Phl p 2, 45.3 ± 0.12% for Phl p 5, 24.7 ± 0.22% for Phl p 6, and 38.3 ± 0.2% for Phl p 1)., Conclusions: Inhibition experiments confirm the hypothesis that SSPs obtained after the osmotic shock of pollen involved in TA, namely grass, pellitory and olive tree pollen, contain allergens and therefore they can induce severe asthma attacks during thunderstorms., (© 2021 John Wiley & Sons Ltd.)

Summary: Objectives. To evaluate the tolerability and efficacy of Olea europaea subcutaneous immunotherapy (SCIT) on patients with rhinoconjunctivitis. Methods. In this open clinical trial patients were assigned to an abbreviated build-up scheme. The outcomes were: number, percentage, and severity of adverse reactions. Secondary outcomes included: changes in immunoglobulin titers and changes in dose-response skin prick tests. Results. Only 8 systemic reactions were registered, which represented 7/47 (14.9%) of patients and 8/429 (1.9%) of administered doses. Regarding immunological parameters the significant increases of sIgG and sIgG4 evidenced the changes in the patient immune system. Cutaneous reactivity decreased significantly. Conclusions. Olea europaea SCIT (Allergovac® depot ROXALL Medicina España S.A.) showed a good safety and tolerability profile. Immunological changes with induction of blocking IgG and decreases in cutaneous reactivity were detected in the patients.

Background and Objectives: The association between pollen counts and allergen levels in the air is controversial. Objectives: The aims of the study were to quantify total and major allergen levels of Phleum pratense and Olea europaea and to analyze their correlation with grass and olive pollen counts and the number of asthma attacks attended at Complejo Hospitalario Universitario, Cáceres, Spain., Material and Methods: A volumetric air sampler and a Burkard spore trap were used for pollen and aeroallergen collection during April- June 2011. Filters were extracted, and major allergens were quantified using enzyme-linked immunosorbent assay., Results: May was the main grass pollination period, with a maximum peak of 1362 grains/m3 (May 13). The main pollination period for olive was April 30-May 20, with a maximum peak of 851 grains/m3 (May 11). A moderate correlation was observed between asthma exacerbations and grass pollen counts or Phleum total allergen levels; this became stronger when a 3-day offset was introduced. A significant association was observed between asthma exacerbations and total olive allergen or olive pollen grain levels when a 1-day offset was introduced. The maximum correlation (moderate-high) was observed 4 days and 6 days away from the maximum olive pollen peak and the maximum Ole e 1 peak level, respectively., Conclusions: This study reveals a significant correlation between grass and olive pollination and an increase in the number of visits to the emergency room for asthma attacks. The aerobiological pattern of allergen levels in the air is similar to that of pollen counts during the grass and olive pollination periods.

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree ( Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli , was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.

Introduction and Objectives: Profilin is a panallergen contained in pollen, plant foods and latex. Although cross-reactivity is expected while performing skin prick tests (SPT) with allergens that contain profilin, this is not always noticed. The purpose of this study was to detect if profilin is contained in the commercial SPT extracts of pollen and plant foods which, in their fresh form, contain determined epitopes of profilin., Material and Methods: Commercial SPT extracts of different pharmaceuticals were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The study included purified palm date profilin, peach (whole, pulp and peel extracts), hazelnut, Olea europea, Parietaria judaica and Phleum pratense., Results: Profilin was detected in all, but peach extracts; it was neither contained in the whole peach extract nor in the ones of peel or pulp., Conclusion: The only accurate way to detect sensitization to profilin, while performing SPT, is the use of purified profilin extract. Even if a plant food or pollen contain an identified molecule of profilin, the relevant SPT commercial extract may not., (Copyright © 2018 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Although plant and fruit pollens are entomophilous and relevant in exposed workers, we have shown a high frequency of sensitisation and symptoms induction of peach tree pollen (PTP) and Prunus persica 9 (Pru p 9) in adults from areas of peach cultivars., Methods: We studied the sensitisation and clinical relevance of PTP and Pru p 9 in a large group of children and adolescents aged 3-19 years. A detailed questionnaire plus skin prick testing to prevalent allergens, PTP, and Pru p 9 were carried out. The clinical relevance was established by nasal provocation test (NPT) and symptom score index., Results: We evaluated 685 children (mean age 8.75 ± 3.3 years, median 9 years), 52% of them female. Sensitisation to PTP occurred in 20% of the cases following olive tree (33%) and Phleum pratense (26%). In a randomly selected subgroup of subjects sensitised to PTP, 30% were skin prick test-positive to Pru p 9. Most cases had rhinitis or rhinoconjunctivitis. NPT showed the relevance of PTP and Pru p 9 in the induction of symptoms., Conclusion: PTP and Pru p 9 are relevant in the induction of sensitisation and respiratory symptoms in children and adolescents. This allergen should be evaluated in children living in regions of peach tree cultivars., (© 2019 S. Karger AG, Basel.)

Aim: To compare the immunological and clinical changes induced by allergen-specific immunotherapy (AIT) using different adjuvants., Materials & Methods: Olea europaea pollen-sensitized mice were treated with olea plus aluminum hydroxide, calcium phosphate, monophosphoryl lipid A (MPL) or immunostimulatory sequences (ISS)., Results: Aluminum hydroxide seems to drive initially to a Th2-type response. Bacteria-derived adjuvants (MPL and ISS) skew the immune response toward Th1 and Treg pathways. Specific-IgE production was lower after AIT with MPL and ISS. Moreover, IgG2a production significantly increased in ISS-treated mice. Bacteria-derived adjuvants also improved the Th1 cytokine response due to IFN-γ higher secretion. In addition, they improved bronchial hyper-reactivity and lung inflammation., Conclusion: Bacteria-derived adjuvants may enhance the efficacy of AIT.

The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen., Significance: Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization using sera from olive pollen allergic patients and cell-based assays, we observed that the recombinant allergen retained the antigenic and allergenic properties of the natural allergen. Collectively, we show that the recombinant protein assembled by proteomics would be suitable for a better in vitro diagnosis of olive pollen allergic patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The overlapping grass and olive pollen seasons in Spain and the phenomenon of cross-reactivity can make it difficult to determine the true causative agent of seasonal allergic rhinitis when only skin prick tests with whole extracts are used. The aim of the GRAMOLE study was to determine sensitization patterns to the major grass and olive pollen allergens detected using specific recombinant IgE and to explore how this knowledge affected physicians' choice of allergen-specific immunotherapy., Methods: Epidemiological, observational, multicenter, cross-sectional study. Results from children under 18 years of age diagnosed with seasonal allergic rhinitis by positive skin prick tests to olive and grass pollen were analyzed. Specific IgE to Phl p 1+5, Ole e 1, and Phl p 7+12 was determined. Investigators specified the optimal composition of allergen immunotherapy before and after knowing the results of the molecular diagnosis., Results: A total of 281 patients with a mean age of 13.4 years were included. Double sensitization to both major allergens was found in vitro in 76% of children for an IgE cutoff point of 0.35 kU/L. When the molecular diagnosis results were known, specialists changed the composition of the prescribed immunotherapy in 52.87% of cases., Conclusions: Double sensitization to grass and olive pollen is common in Spain and also occurs in the pediatric population. Molecular diagnosis using specific IgE may help improve immunotherapy selection in polysensitized patients., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Cross-reactivity reactions between allergenic polygalacturonases (PGs) from different biological sources, especially foods and pollens from the Oleaceae family, have been described using Salsola kali PG (Sal k 6). No PG from olive pollen has been characterized to date, hampering further knowledge about cross-reactions through PGs., Objectives: The aim of this work was to determine the potential allergenicity of the PG from olive pollen and clarify its role in cross-reactivity., Methods: A cDNA-encoding olive pollen PG sequence was subcloned into the pET41b vector and used to transform BL21(DE3) Escherichia coli cells to produce a His-tag fusion recombinant protein. The allergenic properties of olive pollen PG were determined by immunoblotting and ELISA in comparison to Sal k 6. The cross-reactivity potential of the protein with other pollen sources was analyzed by inhibition immunoassays., Results: The existence of other isoforms of Ole e 14 with different allergenicity was confirmed by proteomics and a meta-analysis of the recently reported olive genome. Sal k 6 showed a higher IgE recognition than Ole e 14 regardless of patient sensitization, suggesting the existence of more allergenic Ole e 14 isoforms in olive pollen. IgG and IgE inhibition assays supported the existence of cross-reactions between them and with other PGs from Oleaceae and Poaceae plant families., Conclusions: A new allergen from olive pollen, Ole e 14, has been identified, produced as a recombinant isoform, and structurally and immunologically characterized. Its role in cross-reactivity has been confirmed and, due to its smaller IgE binding capacity, it could have an important role for therapeutic purposes., (© 2018 S. Karger AG, Basel.)

Background: Over the last years, different works have been published about the importance of incorporating new diagnosis techniques in allergic patients such as component-resolved diagnosis (CRD). The objective of this study is to compare the evolution of allergic sensitisation profiles by means of CRD and cutaneous tests (SPT) on pollen-allergic patients., Methods: A total of 123 patients aged between 2 and 14 years were included in an open, prospective, multicentre study. All the children had symptoms suggestive of seasonal respiratory allergic disease, with the diagnosis confirmed by cutaneous tests. Specific-IgE to major pollen-allergens (CRD) and SPT were performed at basal and after three years of follow-up., Results: Out of 123 patients included, a total of 85 were analysed. The mean age was 8±3 years. Significant changes in the allergic sensitisation profiles were observed for the most prevalent allergens (Olea and grass) but it is in grass, the most relevant allergen in terms of allergen pressure, where changes in both absolute and relative frequencies between SPT and CRD were more evident., Conclusion: CRD seems to be an essential tool to carry out an appropriate follow-up of patients with allergic respiratory disease, as well as to decide on the immunotherapy composition that best matches the allergic sensitisation profile of patients., (Copyright © 2016 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs)., Methods: Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA., Results: Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ., Conclusions: Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses., (© 2017 S. Karger AG, Basel.)

Background: Global climate changes may influence the geographical spread of allergenic plants thus causing new allergen challenges., Objective: Allergy patients from two German federal states were compared for their status quo sensitization to ragweed, an establishing allergen, olive, a non-established allergen, and the native allergens birch, mugwort, and ash., Methods: Between 2011 and 2013, 476 adult allergy patients per region were recruited. Patients completed a questionnaire, participated in a medical interview, and underwent skin prick testing and blood withdrawal for analysis of specific IgE to allergen components (ISAC technology). Data on regional pollen load from 2006 to 2011 were acquired from the German Pollen Information Service Foundation., Results: Prick test reactivity to ragweed and ash, respectively, was lower in Bavaria than in NRW (ragweed: p=0.001, aOR=0.54; ash: p=0.001, aOR=0.59), whereas prick test reactivity to olive was higher (p=0.000, aOR=3.09). Prick test reactivity to birch and mugwort, respectively, did not significantly differ. 1% (1/127) of patients with prick test reactivity to ragweed showed sIgE to Amb a 1, and 65% (86/132) of olive-but-not-ash reactive patients showed sIgE to Ole e 1 (NRW: 67%, Bavaria: 65%; p=0.823, OR=0.91). Regional differences in sensitization pattern were neither explainable by cross-reactivity to pollen pan-allergens nor non-exposure variables nor by reported plant population or pollen data., Conclusions: Spread of ragweed and particularly olive may result in prompt occurrence of allergic symptoms. Early identification of invasive allergens due to climate change does need time and spatial close meshed measurement of respective indicator allergens and sensitization pattern., (Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Olive seeds, which are a raw material of interest, have been reported to contain 11S seed storage proteins (SSPs). However, the presence of SSPs such as 7S vicilins has not been studied. In this study, following a search in the olive seed transcriptome, 58 sequences corresponding to 7S vicilins were retrieved. A partial sequence was amplified by PCR from olive seed cDNA and subjected to phylogenetic analysis with other sequences. Structural analysis showed that olive 7S vicilin contains 9 α-helixes and 22 β-sheets. Additionally, 3D structural analysis displayed good superimposition with vicilin models generated from Pistacia and Sesamum. In order to assess potential allergenicity, T and B epitopes present in these proteins were identified by bioinformatic approaches. Different motifs were observed among the species, as well as some species-specific motifs. Finally, expression analysis of vicilins was carried out in protein extracts obtained from seeds of different species, including the olive. Noticeable bands were observed for all species in the 15-75 kDa MW interval, which were compatible with vicilins. The reactivity of the extracts to sera from patients allergic to nuts was also analysed. The findings with regard to the potential use of olive seed as food are discussed.

Background: The identification of the allergenic molecules, associated to the advances in the field of recombinant allergens, led to the development of a new concept in allergy diagnosis called component-resolved diagnosis. The aim of our study was to evaluate the diagnostic accuracy of different allergen components using the full automatic singleplex quantitative platform Immulite™ 2000., Methods: One hundred ninety-five allergic outpatients (35 to olive pollen, 35 to birch pollen, 35 to profilin, 35 to house dust mites, 35 to peach, and 20 to shrimp) and 20 negative controls were enrolled for the study. Bet v 1, Bet v 2, Ole e 1, Der p 1, Der p 2, Der f 1, Der f 2, Pru p 3, tropomyosin were tested both with Immulite™ 2000 and ImmunoCAP™ (Thermo Fisher Scientific, Uppsala, Sweden)., Results: Sensitivity of allergen-specific Immunoglobulin E (sIgE) to Ole e 1, Bet v 1, Der p 1, Der p 2, Der f 1, Der f 2, Pen m 1, and Pru p 3 with Immulite™ 2000 was 100%, 100%, 77.1%, 94.3%, 71.4%, 94%, 75%, and 97.1%, respectively, and the specificity was 100% for all the allergens. The overall agreement between Immulite™ 2000 and ImmunoCAP™ (Thermo Fisher Scientific) platforms was 98.6% (Cohen's kappa = 0.979; confidence interval [CI] 95%: 0.960-0.997). From moderate to strong, positive linear correlations between the assays (r(2) from 0.322 to 0.860, and Spearman's rho from 0.824 to 0.971) were showed., Conclusions: A high diagnostic accuracy of the sIgE to allergen components measurement with Immulite™ 2000 and a high agreement with ImmunoCAP™ platforms were shown in this study., (© 2014 Wiley Periodicals, Inc.)

Olive tree is one of the main allergy sources in Mediterranean countries. The identification of the allergenic repertoire from olive pollen has been essential for the development of rational strategies of standardization, diagnosis, and immunotherapy, all of them focused to increase the life quality of the patients. From its complex allergogram, twelve allergens - Ole e 1 to Ole e 12 - have been identified and characterized to date. Most of them have been cloned and produced as recombinant forms, whose availability have allowed analyzing their three-dimensional structures, mapping their T-cell and B-cell epitopes, and determining the precise allergenic profile of patients for a subsequent patient-tailored immunotherapy. Protein mutant, hypoallergenic derivatives, or recombinant fragments have been also useful experimental tools to analyze the immune recognition of allergens. To test these molecules before using them for clinic purposes, a mouse model of allergic sensitizations has been used. This model has been helpful for assaying different prophylactic approaches based on tolerance induction by intranasal administration of allergens or hypoallergens, used as free or integrated in different delivery systems, and their findings suggest a promising utilization as nasal vaccines. Exosomes - nanovesicles isolated from bronchoalveolar lavage fluid of tolerogenic mice - have shown immunomodulatory properties, being able to protect mice against sensitization to Ole e 1., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related family (PR-5) of plant defense proteins. TLPs from only 32 plant genera have been identified as pollen or food allergens. IgE epitopes on allergens play a central role in food allergy by initiating cross-linking of specific IgE on basophils/mast cells. A comparative analysis of pollen- and food-allergenic TLPs is lacking. The main objective of this investigation was to study the structural and allergenicity features of sapodilla (Manilkara zapota) acidic TLP (TLP 1) by in silico methods. The allergenicity prediction of composite sequence of sapodilla TLP 1 (NCBI B3EWX8.1, G5DC91.1) was performed using FARRP, Allermatch and Evaller web tools. A homology model of the protein was generated using banana TLP template (1Z3Q) by HHPRED-MODELLER. B-cell linear epitope prediction was performed using BCpreds and BepiPred. Sapodilla TLP 1 matched significantly with allergenic TLPs from olive, kiwi, bell pepper and banana. IgE epitope prediction as performed using AlgPred indicated the presence of 2 epitopes (epitope 1: residues 36-48; epitope 2: residues 51-63), and a comprehensive analysis of all allergenic TLPs displayed up to 3 additional epitopes on other TLPs. It can be inferred from these analyses that plant allergenic TLPs generally carry 2-3 IgE epitopes. ClustalX alignments of allergenic TLPs indicate that IgE epitopes 1 and 2 are common in food allergenic TLPs, and IgE epitopes 2 and 3 are common in pollen allergenic TLPs; IgE epitope 2 overlaps with a portion of the thaumatin family signature. The secondary structural elements of TLPs vary markedly in regions 1 and 2 which harbor all the predicted IgE epitopes in all food and pollen TLPs in either of the region. Further, based on the number of IgE epitopes, food TLPs are grouped into rosid and non-rosid clades. The number and distribution of the predicted IgE epitopes among the allergenic TLPs may explain the specificity of food or pollen allergy as well as the varied degree of cross-reactivity among plant foods and/or pollens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The thaumatin-like protein (TLP) Ole e 13 in raw olive fruit is responsible for occupational allergy in olive oil mill workers. However, these workers do not experience allergic symptoms after ingestion of edible olive., Objectives: To analyze the presence of IgE-reactive TLP in raw and edible olive fruit and to assess the allergenic potency of both sources., Methods: The content of TLP in raw and edible olive fruit protein extracts was analyzed using immunoblotting with sera from allergic patients and with olive TLP-specific IgG. The structural and immunological stability of TLP were assayed using immunoblotting after treatment of both raw olive and purified TLP with 0.25 M NaOH solution for 24 hours. Olive pollen extract was investigated by immunoblotting for TLP content., Results: The TLP contained in raw olive fruit was not present in edible olives as a result of maceration before human consumption. No TLP was detected in olive pollen using specific IgG or sera from patients allergic to olive fruit. Sera from patients allergic to olive pollen did not react with purified TLP., Conclusions: IgE-reactive TLP is not present in edible olive, thus explaining the low number of patients allergic to this highly consumed fruit. Patients allergic to olive pollen are not sensitized toTLP and, therefore, not expected to be at risk of food allergy to olive fruit or TLP plant sources.

Isoflavone reductase-like proteins (IRLs) are enzymes with key roles in the metabolism of diverse flavonoids. Last identified olive pollen allergen (Ole e 12) is an IRL relevant for allergy amelioration, since it exhibits high prevalence among atopic patients. The goals of this study are the characterization of (A) the structural-functionality of Ole e 12 with a focus in its catalytic mechanism, and (B) its molecular allergenicity by extensive analysis using different molecular computer-aided approaches covering (1) physicochemical properties and functional-regulatory motifs, (2) sequence analysis, 2-D and 3D structural homology modeling comparative study and molecular docking, (3) conservational and evolutionary analysis, (4) catalytic mechanism modeling, and (5) sequence, structure-docking based B-cell epitopes prediction, while T-cell epitopes were predicted by inhibitory concentration and binding score methods. Structural-based detailed features, phylogenetic and sequences analysis have identified Ole e 12 as phenylcoumaran benzylic ether reductase. A catalytic mechanism has been proposed for Ole e 12 which display Lys133 as one of the conserved residues of the IRLs catalytic tetrad (Asn-Ser-Tyr-Lys). Structure characterization revealed a conserved protein folding among plants IRLs. However, sequence polymorphism significantly affected residues involved in the catalytic pocket structure and environment (cofactor and substrate interaction-recognition). It might also be responsible for IRLs isoforms functionality and regulation, since micro-heterogeneities affected physicochemical and posttranslational motifs. This polymorphism might have large implications for molecular differences in B- and T-cells epitopes of Ole e 12, and its identification may help designing strategies to improve the component-resolving diagnosis and immunotherapy of pollen and food allergy through development of molecular tools.

Molecular evidence on the heterogeneity present in the Ole e 1 allergen of the olive pollen is emerging. Such polymorphism is dependent on the cultivar origin of pollen, which also determines wide differences in the expression of this protein. Determination of biochemical and molecular characteristics of Ole e 1 pollen allergen in two Iranian olive cultivars, namely 'Rowghani' and 'Zard' is necessary to assess their allergenicity potential. SDS-PAGE and immunoblotting analysis of pollen extracts showed that both cultivars present high and low expression of Ole e 1, respectively. These protein levels correlated with similarly different levels of transcripts, as determined by RT-PCR. Two-dimensional protein profiles also showed conspicuous differences in the distribution and the level of expression of those spots reacting to an anti-Ole e 1 antibody. Bioinformatic analysis of four Ole e 1 sequences corresponding to 'Rowghani' and two sequences for 'Zard', showed numerous heterogeneities when compared with those Ole e 1 and Ole e 1-like sequences present in databases. Nucleotide substitutions resulted in many cases in changes over the predicted amino acid sequences. A cladistic analysis of the sequences showed Iranian entries in a central position between West-European sequences, and Ole e 1-like sequences from other Oleaceae species. Moreover, amino acid changes affected key epitopes of the protein involved in the recognition of the protein by the human immune system. Putative implications of polymorphism in both the biological role and the allergic reactivity of Ole e 1 are discussed.

Background and Objective: In areas with a high number of allergens and high allergen concentrations, it is essential to identify the main causes of allergy, especially in pediatric patients. This study was conducted in allergic patients aged 14 or less to identify sensitization profiles during an initial phase, and to then evaluate changes in these profiles after 3 years of follow-up. This article describes the first phase of our investigation., Methods: A total of 187 patients aged between 2 and 14 years were included by 5 allergy units; all the children had symptoms suggestive of allergic disease (rhinoconjunctivitis andlor asthma). Allergy diagnosis was confirmed by evaluation of clinical history, allergen exposure, and in vivo or in vitro tests. Specific immunoglobulin E (slgE) to major allergens was tested., Results: Patients were sensitized to both seasonal (especially grass, olive, cypress and Cynodon dactylon) and perennial allergens (Alternaria alternata) and to panallergens (especially profilin and lipid transfer protein). Almost 60% of the patients included were polysensitized. Sensitization to certain major allergens such as Cup s1, Phl p1, or Sal k1 seems to increase with age. Patients sensitized to profilin had a higher number of sensitizations than non-profilin-sensitized patients. This panallergen is a diagnostic confounding factor., Conclusions: A high percentage of allergic pediatric patients living in an area with high exposure levels to a large number of allergens are polysensitized and have a high percentage of sensitization to panallergens. The implementation of new diagnostic tools such as component-resolved diagnosis is crucial.

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Besides this, some cases of contact dermatitis and food allergy to the olive fruit and olive oil have been also described. Several scientific studies dealing with olive allergens has been reported, being the information available about them constantly increasing. Up to date, twelve allergens have been identified in olive pollen while just one allergen has been identified in olive fruit. This review article describes considerations about allergen extraction and production, also describing the different methodologies employed in the physicochemical and immunological characterization of olive allergens. Finally, a revision of the most relevant studies in the analysis of both olive pollen and olive fruit allergens is carried out., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Polysensitization is common in patients with allergic rhinitis (AR) and may affect clinical feature. However, there are patients who remain monosensitized., Objective: This cross-sectional study aimed at evaluating a large cohort of AR patients to define the percentage and the features of mono- and poly-sensitized subjects., Methods: This observational cross-sectional study included a large group of AR patients: 2415 subjects (1958 males, mean age 24.6 ± 5 years) were consecutively evaluated. Symptom severity, type and number of sensitizations, and AR duration were considered., Results: 621 patients (25.7%) were monosensitized: 377 to Parietaria, 194 to house dust mites, 19 to birch, 17 to grasses, 12 to molds, 2 to olive, and 1 to cypress. There was no difference between mono- and polysensitized patients concerning the duration of rhinitis (6 ± 2.14 years vs 6 ± 3.7). Severity of symptoms was higher in polysensitized patients than in monosensitized (p<0.05); in addition, there was a difference among monosensitized patients: Parietaria-allergy induces the most severe symptoms., Conclusion: This study conducted in a large AR population might suggest that monosensitized and polysensitized AR patients could constitute two different categories. In addition, the specific type of allergy may condition the clinical feature., (Copyright © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Objective: We evaluated, by means of a multicentre, prospective observational trial, the severity in term of symptoms and symptomatic drugs use and the presence of asthma in subjects with tree or cypress or olive, or ragweed or parietaria allergy and we evaluated also the efficacy of a consecutive 2-year specific sublingual immunotherapy treatment., Patients and Method: Consecutive patients suffering of respiratory allergies (rhinoconjunctivitis and/or mild moderate asthma) due to one of the described allergens were enrolled During the specific relevant pollen seasons for each allergens nose and eye symptoms and medication scores (SS and MS) were evaluated Global score (GS) was calculated as the sum of SS and MS. An Asthma symptom score if present, were also, calculated A total of 162 patients were enrolled in 14 Italian Allergic Clinics .Patients were treated with the relevant specific sublingual immunotherapy (SLITOne, ALK) for two consecutive pollen seasons., Results: At baseline prevalence of allergies was the following: tree 18% (30 patients); ragweed 14% (23 patients); olive 7% (11 patients); cypress 7% (12 patients) and pellitory 53% (86 patients). At baseline asthma was detected in 65 patients (40%). Asthma was more common in poli-sensitive subjects in comparison with mono-sensitive (51% vs. 300/o). According to allergen type, asthma was present in 47% of pellitory allergic patients, in 45% of olive allergic subjects, in 38% of ragweed allergic patients, in 26% of tree allergic patients and only in 9% of cypress allergic subjects. In pellitory, olive and ragweed allergic patients the frequency of asthma was statistically significant (P=0.0055) higher in comparison with other groups. At baseline GS mean (SD) in the whole population was 17(7). GS during the following 2 consecutive seasons with SLIT treatment decreased significantly (P=0.0001) to 9 (5) and to 7 (5), respectively (a reduction of 59% in comparison with baseline). In patients with asthma the mean clinical score decreased significantly from baseline value of 2. 7 to 0.3 at the end of the observation period No serious adverse events were reported Local side effects, mainly oral itching, were reported by 14% of patients and were mild and transient in nature., Conclusion: In this population pellitory, olive and ragweed allergies are associated with a more severe clinical picture in comparison with tree and cypress allergy. A two-year SLIT treatment was associated with a significant reduction in SS, MS, GS and asthma score in comparison with baseline. SLIT was also safe and well tolerated

Background: Allergic symptoms are commonly related to atmospheric pollen counts in sensitized allergic individuals. However, concordance between symptoms, pollen counts, and aeroallergen concentrations is not always good., Objectives: To determine the correlation between olive pollen counts, aeroallergen levels, and clinical symptoms in patients with allergic asthma or rhinitis in Ciudad Real (Spain)., Methods: Two types of samplers were used to determine pollen exposure: a Burkard spore trap to collect pollen grains and a high-volume air sampler to collect airborne particles. A total of 366 air filters were collected. After extraction, they were analyzed by specific immunoglobulin E enzyme-linked immunosorbent assay inhibition using a serum pool containing high titers of olive-specific immunoglobulin E. Twenty olive-pollen monosensitized patients were asked to record their daily symptoms before, during, and after the olive pollen season., Results: Olive pollen was detected between April 21 and June 30, 2004. Symptoms showed positive and significant correlations with pollen counts (r = 0.700, P < .001) and aeroallergen levels (r = 0.803, P < .001). Using a Poisson regression model, relative changes in aeroallergen concentrations and pollen counts were found to be similar and significant. Threshold levels for the induction of symptoms were 162 olive pollen grains/m(3) and 22.7 ng of olive pollen allergen/m(3) (equivalent to 0.9 ng/m(3) of Ole e 1)., Conclusions: Olive aeroallergen concentrations and pollen counts are positively associated with symptoms of rhinitis and asthma in olive-allergic patients. Both data may be used in the clinical follow-up of these patients., (Copyright © 2011 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Patients with pollinosis show allergic symptoms related to airborne pollen levels, although this association is not always close. The use of new diagnostic techniques could improve our knowledge of this relationship., Objective: To evaluate the relationship between pollen counts and the results obtained using 2 diagnostic techniques: the skin prick test and allergen-specific immunoglobulin E (slgE) concentrations in serum., Methods: Sixty-eight pollen-allergic patients were diagnosed using a combination of the high-capacity screening approach ADVIA Centaur with a panel of 13 purified allergens and a skin prick test (SPT) with conventional extracts. Pollen levels were obtained by means of a volumetric sampler., Results: The highest percentages of sensitization were detected for grass mixture allergens and major recombinant grass allergens (Phl p 1 and Phi p 5), followed by olive tree extracts and olive allergens (Ole e 1 and Ole e 9), in SPT and using recombinant allergens, respectively. The main pollen types registered in the atmosphere during 2006 and 2007 were Quercus, Poaceae, and Cupressaceae. A statistically significant correlation was observed between total pollen levels and median values of slgE, especially in 2007., Conclusion: A strong and significant positive correlation was found between pollen counts and slgE levels. This correlation was weaker in the case of SPT and airborne pollen.

Background: Navarre, in Northern Spain, is an area with moderate exposure to olive and ash tree pollen., Objective: To assess the relevance of ash as a cause of pollinosis in our region., Methods: The study sample comprised 85 patients from Navarre with clinical symptoms of pollinosis. Specific immunoglobulin E (sIgE) was determined to Fra e 1, Ole e 1, and a mixture of amino- and carboxy-terminal domains of Ole e 9 (Ole e 9 NC) (ADVIA-Centaur). At the same time, the presence of sIgE to other pollen allergens was studied. Prick tests were performed with ash pollen (n=33) and olive pollen (n=85) and the symptomatic period was recorded (n=85). As a control group, we studied the serum of 98 patients with olive pollen allergy, intense exposure to olive pollen, and no exposure to ash., Results: Sensitization to Oleaceae was detected in 24/85 patients in the study group (28.2%). In this group, the mean (SD) level of IgE to Fra e 1 was 8.5 (10) kU(A)/L and to Ole e 16.07 (7.88) kU(A)/L (P < .001). In the control group, these figures were 103.64 (132.19) kU(A)/L and 86.43 (118.5) kU(A)/L (P < .001), respectively. In all patients with positive sIgE to Fra e 1, IgE to Ole e 1 was also detected (concordance index, kappa = 1), both in the study group and in the control group. Patients who were sensitized to Fra e 1 did not present a longer symptomatic period and their symptoms did not have an earlier onset., Conclusion: We did not find evidence of clinically relevant sensitization to ash in Navarre.

Background: Cross-reactivity among plant food allergens belonging to the nonspecific lipid transfer protein (LTP) family is well known. In contrast, the relationship among these allergens and their putative homologs from olive (Ole e 7) and Parietaria (Par j 1) pollen has not been clarified., Methods: Sera with specific IgE to LTP allergens were obtained from peach-, mustard- and olive pollen-allergic patients. Purified LTP allergens from foods (peach, apple, mustard and wheat) and pollens (olive, mugwort and Parietaria) were tested by ELISA and ELISA-inhibition assays., Results: Plant food LTP-allergic patients showed a significantly higher number of sera (89-100 vs. 33-64%) with specific IgE and mean specific IgE levels (0.30-1.56 vs. 0.21-0.34 OD units) to the 4 food LTP allergens tested than to olive Ole e 7 and Parietaria Par j 1 pollen. ELISA-inhibition assays indicated cross-inhibition between food LTP allergens but no cross-reactivity between these allergens and Ole e 7 and Par j 1, or, even more, between the LTP allergens from olive and Parietaria pollen., Conclusions: LTP allergens from olive and Parietaria pollen cross-react neither with allergenic LTPs from plant foods nor between themselves. Therefore, both pollens do not seem to be related with the LTP syndrome., (Copyright © 2011 S. Karger AG, Basel.)

Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%alpha-helix, 50%beta-sheet and 27%beta-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.

Background: Subcutaneous immunotherapy is an etiological therapy for certain IgE-mediated diseases. It is usually administered in 2 phases: induction and maintenance. Administration in clustered schedules during the induction phase may be a valid alternative to reach the maintenance dose early if the treatment is well tolerated., Objectives: To compare the tolerability of different clustered schedules in subcutaneous immunotherapy with standardized allergen extracts administered and to identify factors associated with increased risk of systemic reactions (SRs)., Methods: Retrospective, observational, multicenter study in patients with allergic respiratory disease., Results: Data from 1,147 patients were collected. Thirty-nine patients (3.4%) experienced 42 SRs (0.6% of doses). According to the European Academy of Allergy and Clinical Immunology SR grading system, there were 7 grade 0 reactions (16.7%), 26 grade 1 reactions (61.9%), 8 grade 2 reactions (19.0%), and 1 grade 3 reaction (2.4%). There were no grade 4 SRs (anaphylactic shock). We observed a higher risk of SRs in patients who received an initial dose higher than 0.3 index of reactivity (IR); only 2 reactions occurred after administration of the initial dose of the regimen, both with 0.4 IR. The remainder appeared in subsequent injections, although never with a dose lower than 0.35 IR., Conclusions: Clustered regimens with IR-standardized extracts are an alternative to classic immunotherapy thanks to their low incidence of SRs compared with other rapid regimens during the induction phase. The ideal clustered regimen should start at an initial dose no greater than 0.35 IR to minimize the incidence of SRs.

Background: Nasal polyposis is highly prevalent in the general population. Its exact origin is unknown, although several factors are involved in the etiology and development of this condition. Clinical patterns, a history of atopy, environmental exposure, eosinophil-mediated inflammation, the presence of inflammatory mediators, and sensitization to some allergens indicate that nasal polyposis is associated with allergic phenomena. The aim of this study was to identify the association between nasal polyposis and allergic factors by examining hypersensitivity reactions to common allergens and environmental exposure that could lead to the development of atopy., Methods: We conducted a comparative study of 190 patients with nasal polyposis and 190 healthy individuals. The study included clinical and epidemiological variables, environmental exposure factors, and an allergology workup using skin prick tests with 18 inhaled allergens., Results: A total of 121 patients (63.7%) of the 190 were male; 62.1% had a family history of allergy. The incidence of asthma was 48.9% among the patients and only 2.3% among the controls (P < .001). The factor most frequently involved in the patients' symptoms was weather changes (67.4%). Skin prick tests were positive in 63.2% of the patients and 31.1% of the controls. The allergens that most frequently elicited a reaction from the patients in the prick tests were Dermatophagoides pteronyssinus (27.7%), Dermatophagoides farinae (21.3%), and Olea europaea (21.1%). The difference between these results and those of the controls was statistically significant., Conclusions: Patients with nasal polyposis are sensitive to the most common allergens in our environment and exhibit a clear-cut correlation with other allergic factors, as confirmed by personal and family histories, the presence of chronic rhinitis, and the results of in vivo tests.

Background: Allergy diagnosis in patients exposed to multiple pollen species is complex and misdiagnosis is often a cause for unsuccessful specific immunotherapy., Objective: We studied the sensitization profile of individual allergens (major, minor and pan-allergens) in pollen-sensitized patients in a region with high exposure to olive pollen by investigating the influence of minor allergens on allergic disease and the association between pan- and minor allergen sensitizations., Methods: A panel of 13 purified allergens, which included the most relevant allergens in the area, as well as minor olive allergens and pan-allergens, were screened using a high-capacity technology (ADVIA-Centaur) in 891 patients., Results: Olive allergy as measured by specific IgE to Ole e 1 was the leading pollinosis in the area. The minor olive allergens Ole e 7 and Ole e 9 were markers of more severe allergic illness. Profilin sensitization was associated mainly with grass allergy, the second most prevalent pollinosis. Salsola kali pollen allergy was the third most common cause of pollinosis in the area. The prevalence of sensitization to the peach allergen Pru p 3, a nonspecific lipid-transfer protein, was notable., Conclusion: Epidemiological analysis by component-resolved diagnosis is a new method, which elucidates the interaction between allergen exposure gradient and patient sensitization. High exposure leads to differential sensitization profiles some of which are associated with more severe allergic conditions. Profilin sensitization, related mainly to grass pollinosis, was a marker of more severe grass pollen sensitization.

Background: Numerous varieties of Olea europaea have been described in Mediterranean countries., Objective: To investigate the immunochemical characteristics of 6 varieties of Olea europaea collected during 5 consecutive years., Methods: The varieties Carrasquefio, Manzanillo, Acebuche (wild olive), Hojiblanco, Picual, and Nevado were analyzed. Pollen samples from each variety were collected for 5 consecutive years from the same cultivars by trained personnel. The antigenic and allergenic profiles of these extracts were evaluated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using the serum of 29 O. europaea-allergic individuals. Ole e 1 was measured using enzyme-linked immunosorbent assay and purified Ole e 1 and rabbit polyclonal antibodies. Allergenic potency was determined using enzyme-linked immunosorbent assay inhibition and is expressed in histamine equivalent prick units per gram of raw material., Results: Hojiblanco and Acebuche had the lowest mean +/- SD Ole e 1 content in the 5 years (0.045 +/- 0.029 and 0.059 +/-0.031 microg/microg of freeze-dried material, respectively). The variety with the highest mean +/- SD Ole e 1 content was Picual (0.19 +/-0.075 microg/microg). Hojiblanco had the lowest total biological potency throughout the study. A positive correlation was obtained between rainfall in the winter months and total allergenicity of the 6 varieties., Conclusions: The different varieties of O. europaea pollen demonstrated great differences in allergenic potency and Ole e 1 content. These differences were maintained throughout the study, suggesting that they are due to genetic differences intrinsic to the varieties, although certain climatic effects may also play a role.

This article summarizes the most important advances of recent years in the field of gene-environment interaction in allergic response. It specifically examines sensitization to olive pollen as an example of one of the main causes of allergic disease in the Mediterranean area. The presence of at least 20 proteins with allergic activity has been demonstrated in olive pollen, and 10 of these have been characterized (Ole e 1 to Ole e 10). Ole e 1, which is considered to be the majority allergen (causing sensitization in more than 70% of patients), has been the subject of many studies looking for risk factors and ways to protect against sensitization. Markers of the major histocompatibility complex and other genetic loci associated with the allergic response have been analyzed using population-based, family-based, and functional approaches, which have revealed the involvement of genetic regulation in this type of response. Furthermore, evaluation of environmental factors and their relationship with genetic factors is essential when attempting to understand this type of disease. In this review, we provide examples of how exposure to high doses of olive pollen allergen in a specific genetic context can trigger different allergic conditions (from asthma to nonresponse). We stress the importance of evaluating these factors in order to modulate this response correctly.

This study analyzes the influence of the IgE response to certain olive pollen allergens in the modulation of the different clinical phenotypes of allergic disease and their relationship with the level of exposure to pollen and genetic factors. Patients from high-exposure areas had a complex IgE antibody response to allergens of Olea euroapea, which included 3 or more allergens in 75% of cases. The majority allergens were Ole e 1, Ole e 2 (profilin), Ole e 7 (lipid transporting protein), Ole e 9 (glucanase), and Ole e 10. The existence of the antigen HLA-DR2 (15) led to a higher risk of sensitization to Ole e 10 and a greater trend towards the development of severe asthma, which increased in the presence of an anti-profilin IgE. Thirty percent of patients suffering from pollinosis simultaneously presented allergy to vegetable foods. Anti-Ole e 7 IgE was significantly associated with fruit anaphylaxis and anti-profilin IgE was detected in 90% of patients with oral syndrome. Finally, we analyzed the role of glucanase and Ole e 10 as causes of the pollen-latex-fruit syndrome.

Allergen-specific immunotherapy (SIT) is the only allergen-specific treatment for allergy. It can prevent progression of the disease and has a long-lasting therapeutic effect. Since SIT is allergen-specific, the identification of the disease-eliciting allergen is an essential prerequisite for the accurate prescription of treatment. Diagnostic tests based on allergen extracts consist of mixtures of various allergens of which some are specific for the allergen source and others occur as cross-reactive allergens in various unrelated allergen sources. It may therefore be difficult and sometimes impossible to identify the disease-causing allergen with such tests, particularly in patients who are sensitized to more than one allergen source. Sensitization to pollens from olive, grasses, and Parietaria in the Mediterranean area is frequently treated with SIT. Here, we describe allergen molecules from these sources that can be used for component-resolved diagnosis of allergy to facilitate the selection of patients for SIT and monitor the immunological effects of treatment.

The olive tree is grown in many parts of the world. Its germplasm is very broad, with 250 varieties in Spain alone. Variations in the ability of pollen to germinate have been studied in detail and show conspicuous differences between varieties. However, commercial olive pollen from cultivars whose origin is unknown is the material that is commonly used for clinical and biological studies. We aim to assess the putative heterogeneity of olive cultivars with regard to the presence of several pollen allergens and to determine whether these differences have biological and clinical relevance. Previous studies show that most allergens isolated and characterized to date are highly polymorphic. Olive cultivars display wide differences in the expression levels of many allergens and in the number and molecular characteristics of the allergen isoforms expressed. These differences are maintained over the years, and are intrinsic to the genetics of each cultivar. Such broad polymorphism seems to be involved in the physiology of the olive reproductive system, which might include the adaptation of the plant to different environmental conditions, the establishment of the compatibility system, and pollen performance. The differences in allergen composition in cultivars, particularly in the Ole e 1 allergen, are responsible for the important differences in the allergenic potency of the extracts. These findings could have a number of implications for the diagnosis and therapy of olive pollen allergy. We discuss how cultivar differences affect extract quality, diagnostic and therapeutic efficacy and safety, and the development of new vaccines based on the use of recombinant allergens.

Background: Very high levels of exposure to olive pollen in the south of Spain lead to differential allergen sensitization profiles. Therefore, new approaches to allergen standardization, diagnosis, and vaccination are necessary., Methods: Quantification of minor allergens in extracts, component-resolved patient diagnosis, and IgG4 individual allergen responses were used to evaluate new strategies in the management of olive pollen allergy., Results: Allergen variability observed between different olive cultivars can be used to identify suitable allergen sources that can be combined to yield consistent allergen extracts for diagnosis and immunotherapy. Component-resolved diagnosis can provide a better patient classification. IgG4 levels to major allergens increase significantly, whereas specific IgG4 to minor allergens does not seem to increase, at least during the early phases of immunotherapy., Conclusion: Patients exposed to extreme olive pollen levels display a different severity of allergy from those exposed to normal levels, which makes it necessary to follow a different clinical approach. The use of component-resolved diagnosis, better standardized allergen extracts, and new efficacy monitoring techniques will lead to a significant improvement in the management of olive allergy disease.

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

Background: English plantain (Plantago lanceolata L.) and olive (Olea europaea L.) pollens are important causes of pollinosis in large areas of North America, Australia, and the Mediterranean basin. The major pollen allergens of both plants, Pla I 1 and Ole e 1, share 38.7% of their amino acid sequences., Objective: To analyze putative cross-reactivity between these 2 proteins., Methods: Several antibodies and patients' sera were used in immunoblot and immunocytochemistry experiments., Results: Two anti-Pla I 1 antibodies were able to bind to 3 polypeptides from olive pollen protein extracts, which correspond to the 3 glycosylation isoforms of Ole e 1 (18-22 kDa) previously described. Moreover, Pla I 1 protein was found in the cytoplasm of both the vegetative and the generative cells of P lanceolata mature pollen. On olive pollen sections, these anti-Pla I 1 antibodies displayed significant labeling in the cytoplasm of the vegetative cell and in both the exine and the material adhering to this outer layer of the pollen wall. In addition, the anti-Ole e 1 antibody 10H1 was found to cross-react with proteins of similar masses (16-20 kDa) to Pla I 1 variants. In Plantago pollen sections, the 10H1 antibody recognized proteins located in the cytoplasm of both the vegetative and generative cells. Cross-reaction was confirmed using sera from patients allergic to either plant pollen., Conclusion: Both allergens share common epitopes, which can be cross-recognized by different antibodies and sera from different patients, although this antigenic similarity seems to have little clinical relevance.

Employing transgenic plants as alternative systems to the conventional Escherichia coli, Pichia pastoris or baculovirus hosts to produce recombinant allergens may offer the possibility of having available edible vaccines in the near future. In this study, two EF-hand-type Ca2+-binding allergens from olive pollen, Ole e 3 and Ole e 8, were produced in transgenic Arabidopsis thaliana plants. The corresponding cDNAs, under the control of the constitutive CaMV 35S promoter, were stably incorporated into the Arabidopsis genome and encoded recombinant proteins, AtOle e 3 and AtOle e 8, which exhibited the molecular properties (i.e. MS analyses and CD spectra) of their olive and/or E. coli counterparts. Calcium-binding assays, which were carried out to assess the biochemical activity of AtOle e 3 and AtOle e 8, gave positive results. In addition, their mobilities on SDS/PAGE were according to the conformational changes derived from their Ca2+-binding capability. The immunological behaviour of Arabidopsis-expressed proteins was equivalent to that of the natural- and/or E. coli-derived allergens, as shown by their ability to bind allergen-specific rabbit IgG antiserum and IgE from sensitized patients. These results indicate that transgenic plants constitute a valid alternative to obtain allergens with structural and immunological integrity not only for scaling up production, but also to develop new kind of vaccines for human utilization.

Background: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease., Objective: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis., Methods: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays., Results: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively., Conclusions: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

Background: Allergy to latex of Hevea brasiliensis is a frequent problem. In spite of the significant progress of research, the identity and cross-reactivity of some latex allergens are unknown., Objective: To identify, among the fully characterized latex proteins, those with a higher probability to be allergenic., Methods: We used in silico techniques (amino acid sequence comparison and molecular modelling) to identify potential new allergens among the known proteins of H. brasiliensis., Results: Cu/Zn superoxide dismutase, heat shock protein and calmodulin of H. brasiliensis show highly significant (E < 10(-9)) amino acid sequence homologies with known allergens., Conclusion: On the basis of our data, Cu/Zn superoxide dismutase, heat shock protein and calmodulin are the most probable allergens among fully characterized proteins of H. brasiliensis, and could potentially explain, at least in part, the multiple cross-reactivities of latex with vegetable foods and other plant-derived products. Consequently, we think that the above proteins should be particularly considered in the future laboratory and clinical research.