Search

Background Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about allergens implicated in hypersensitivity reactions to these seeds. The present work aimed to identify the allergens involved in IgE-mediated reactions in five patients with a clinical history of severe systemic symptoms after flaxseed consumption. Methods Proteins susceptible to be allergens with IgE-binding capacity were purified from flaxseed extract by chromatographic techniques. Their identification was achieved via MALDI-TOF mass spectrometry. Immunoassays were performed using the five allergic patient's era either by testing them individually or as a pool. Results Proteins susceptible to be allergens with IgE-binding capacity were purified from flaxseed extract by chromatographic techniques. Their identification was achieved via MALDI-TOF mass spectrometry. Immunoassays were performed using the five allergic patient's era either by testing them individually or as a pool. Results: Four out of five patients recognized a low-molecular-mass protein (around 13kDa) by immunoblotting of the flaxseed extract, while two patients recognized a protein of approximately 55 kDa. They were identified by mass spectrometry as flaxseed 2S albumin, included into WHO/IUIS allergen nomenclature as Lin u 1,and 11S globulin, respectively. Inhibition assays revealed in vitro IgE-cross-reactivity of Lin u 1 with peanut and cashew nut proteins, while IgE recognition of 11S globulin by patients' sera was partially inhibited by several plant-derived sources. Conclusions Seed storage proteins from flaxseed were involved in the development of severe symptoms in five individuals and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, it is the first time that the 11S globulin is identified as a potential allergen. We consider that these data should be taken into account for a more accurate diagnosis of patients.

Maria Luisa Somoza1 *, Natalia Pérez-Sánchez2 *, Isabel Torres-Rojas,1 Laura Martín-Pedraza,3 Natalia Blanca-López,1 Laura Victorio Puche,4 Eva Abel Fernández González,5 José Damián López Sánchez,6 Javier Fernández-Sánchez,7,8 Enrique Fernández-Caldas,5,9 Mayte Villalba,10 Francisco Javier Ruano,1 José Antonio Cornejo-García11 *, Gabriela Canto1,12 *, Miguel Blanca3 &#x002A1Allergy Department, Infanta Leonor University Hospital, Madrid, Spain; 2Allergy Department, Hospital Regional Universitario de Málaga, Málaga-IBIMA (FIMABIS), Málaga, Spain; 3Allergy Department, Fundación para la Investigación e Innovación Biomédica (FIIB) de los Hospitales Universitarios Infanta Leonor y Sureste, Madrid, Spain; 4Allergy Department, Morales Meseguer General University Hospital, Murcia, Spain; 5R&D Department, Inmunotek Laboratories, Madrid, Spain; 6Allergy Department, Virgen de la Arrixaca Clinical University Hospital, Murcia, Spain; 7Allergy Department, General University Hospital of Alicante- ISABIAL, Alicante, Spain; 8Clinical Medicine Department, Miguel Hernandez University, Alicante, Spain; 9Division of Allergy and Immunology, University of South Florida College of Medicine, Tampa, FL, USA; 10Department of Biochemistry and Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain; 11Instituto de Investigación Biomédica de Málaga-IBIMA (FIMABIS), Málaga, Spain; 12School of Medicine, Universidad Complutense de Madrid, Madrid, Spain*These authors contributed equally to this workCorrespondence: Maria Luisa Somoza, Email mlsomoza@yahoo.comBackground: Allergy can start at early ages, with genetic and environmental factors contributing to its development.Aim: The study aimed to describe the pattern of sensitisation and allergy in children and adolescents of Spanish versus Moroccan ancestry but born in the same rural area of Spain.Methods: Participants were children and adolescents (3– 19 years) of Spanish or Moroccan descent, born in Blanca, Murcia (Spain). A detailed questionnaire was completed, and skin prick tests were performed to assess reactions to the most prevalent pollen allergens (O. europaea, P. pratense, S. kali, C. arizonica, P. acerifolia, A. vulgaris and P. judaica) plus molecular components Ole e 1 and Ole e 7. The association with ancestry was verified by studying participants’ parents.Results: The study included 693 participants: 48% were aged 3– 9 years and 52%, 10– 19 years; 80% were of Spanish descent and 20% of Moroccan descent. Sensitisation to Olea europaea, Phleum pratense, Salsola kali and Cupressus arizonica were slightly higher in the Spanish group. The only significant differences were observed in sensitisation to Ole e 1 (p=0.02). Rhinitis, conjunctivitis, and rhinitis plus asthma were significantly higher in the Spanish group (p=0.03, p=0.02, p=0.007, respectively). The sensitisation pattern differed between Spanish and Moroccan parents, and between Moroccan parents and their children, but not between Spanish parents and their children.Conclusion: Both environment and ancestry may influence sensitisation and symptoms. Although the environment seems to have a stronger influence, other factors may contribute to the differences in prevalence and in the clinical entities in people of Spanish versus Moroccan descent.Keywords: ancestry, pollen sensitisation, children/adolescents, olive tree pollen, molecular components, respiratory allergy

Objectives: To determine the usefulness of the in vitro and in vivo methods used in the diagnosis of kiwifruit allergy and to specifically assess the impact of seed proteins on sensitivity. Methods: We performed skin prick tests (SPTs) using various commercial extracts, homemade pulp, and seed extracts and prick-prick tests with kiwifruit on 36 allergic patients. The presence of specific IgE (sIgE) was assessed using the ImmunoCAP (kiwifruit extract), ELISA (Act d 1, Act d 2), ISAC, and FABER assays. Immunoblotting of seed extract was carried out, and a single-blind oral food challenge was performed with whole seeds in seed-sensitized individuals. Results: The prick prick test with kiwifruit demonstrated the highest diagnostic capacity (81.8% sensitivity and 94.1% specificity) among the in vivo tests. The sIgE levels measured using ImmunoCAP (kiwifruit extract) showed a similar sensitivity to that of global ISAC and FABER (63.9%, 59.5%, and 58.3%, respectively). Act d 1 was the major allergen. Sensitization to Act d 1 was associated with positive sIgE results to whole kiwifruit extract detected by ImmunoCAP (P

Thematic cooperative health research networks (RETICS) are organizational structures promoted by the Instituto de Salud Carlos III of the Spanish Ministry of Science with the objective of carrying out cooperative research projects addressing challenges of general interest for society as a whole in the field of health care. The RETICS of Asthma, Adverse Drug Reactions, and Allergy (ARADyAL) received funding in 2016 for a 5-year program (2017-2021). ARADyAL integrates basic and clinical research in the areas of allergy, immunology, genetics, nanomedicine, pharmacology, and chemistry, with special interest in research on new biomarkers and the design and evaluation of new interventions for allergic patients with severe phenotypes. The consortium comprises 28 groups across Spain, including 171 clinical and basic researchers, 17 clinical groups that cover more than 10 000 000 patients of all ages from urban and rural areas and 11 basic groups active mostly at universities and research institutes. ARADyAL has proposed a research program organized into 3 different areas focusing on precision medicine, as follows: Program 1, Mechanisms and prediction of adverse drug reactions and allergic diseases; Program 2, Toward a precise diagnosis of allergic diseases; and Program 3, Predicting interventions in allergic diseases. There is also 1 common program dedicated to training. The network has a Steering Committee and an External Advisory Scientific Committee, which advise the global network coordinator, who has recognized expertise in the field. ARADyAL is a unique meeting point for clinicians and basic scientists who are already working in allergy.

A disposable electrochemical PCR-free biosensor for the selective detection of a fragment encoding the protein Sin a 1, a 2S albumin considered a diagnostic marker for sensitization to mustard, is reported. The methodology is based on the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sin a 1 allergen coding sequence with appropriately designed RNA probes. Labeling with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with horseradish peroxidase (HRP) was carried out onto the surface of magnetic beads (MBs). Amperometric transduction was undertaken on screen-printed electrodes using H2O2 as enzyme substrate and hydroquinone (HQ) a redox mediator. The electrochemical biosensor allows the simple and fast detection (75 min) of Sin a 1 reaching a limit of detection of 3 pM. The bioplatform was successfully applied to the analysis of the targeted Sin a 1 gene specific region using just 50 ng of non-fragmented denatured genomic DNA extracted from yellow mustard seeds.

ObjectivesSeveral studies have described peach tree (PT) as an occupational allergen. The aim of this work was to assess the effect ofPrunus persica9 (Pru p 9), a recently identified allergen from PT pollen, in exposed workers.MethodsThe study included people who reported respiratory symptoms after handling PT in orchards during the flowering period (Blanca village, Murcia region, south-east Spain). After completing a detailed questionnaire, participants underwent skin prick test (SPT) and nasal provocation test (NPT). The IgE response was analysed by SDS-PAGE and immunoblotting assays.ResultsA total of 21 cases were included (mean age 45 years; 57% women). Most were polysensitised to common pollens, although one person was sensitised only to PT pollen. All cases had a positive SPT to this pollen, and 43% also to Pru p 9. All participants reported having rhinitis, and six participants reported having also asthma. Immunoblotting showed a heterogeneous IgE pattern for several proteins, with Pru p 9 recognised in nine cases. Most participants sensitised to PT pollen and Pru p 9 had positive NPTs, while those who were not sensitised to Pru p 9 tested negative.ConclusionsWe demonstrate for the first time that Pru p 9, an allergen from PT pollen, can induce respiratory symptoms following occupational exposure. This must be considered a relevant allergen when people working with PT cultivars develop respiratory symptoms.

37 p.-5 fig.-1 tab., 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., This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional (FEDER): SAF2014-53209-R to M.V. and R.B. and SAF2017-86483-R to M.V. from theMinisterio de Economı́a y Competitividad and by the Thematic Networks and Co-operative Research Centres:RIRAAF Network RD12/0013/0015; and ARADyAL(RD16/0006/0013 and RD16/0006/0014) from the Instituto de Salud Carlos III (ISCIII). B.R.-L., A.J., A.N, and C.M.acknowledge PI-01119-2016 from the Consejerı́a de Salud(Junta de Andalucı́a) and the Alergosur Foundation. R.B. also acknowledges the financial support of the PI17CIII/00045grant from the AES-ISCIII program.

The characterization of the humoral response in Alzheimer’s disease (AD) patients might aid in detecting the disease at early stages. We have combined phage display and protein microarrays to identify AD autoantibodies and their target biomarkers. After enrichment of the T7 phage display libraries from AD and healthy brain tissue mRNA in AD-specific phages, 1536 monoclonal phages were printed on microarrays to probe them with 8 AD and 8 healthy control sera. A total of 57 phages showed higher seroreactivity in AD. In total, 13 out of the 44 unique sequences displayed on the phages were selected for validation using 68 AD and 52 healthy control sera. Peptides from Anthrax toxin receptor 1, Nuclear protein 1, Glycogen phosphorylase, and Olfactory receptor 8J1 expressed in bacteria as HaloTag fusion proteins showed a statistically significant ability to discriminate between AD patients and controls. The identified panel of AD autoantibodies might provide new insights into the blood-based diagnosis of the disease., This work was supported by Grants SAF2014-53209-R to M.V. and R.B., Grant PI17CIII/00045 from the AES-ISCIII program to R.B., and Instituto de Salud Carlos III (ISCIII) cofounded by Fondo Europeo de Desarrollo Regional−FEDER for the Thematic Networks and Co-operative Research Centres: RIRAAF Network RD12/0013/0015 and ARADyAL (Grant RD16/0006/0014). We also gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for Grants FIS PI17/01930 and CB16/12/00400. Fundacioń Solorzano Grant FS/38-2017 to M.F. The Proteomics Unit ́ belongs to ProteoRed, PRB3-ISCIII, supported by Grant PT17/0019/0023, of the PE I + D + I 2017-2020, funded by ISCIII and FEDER. P.S.S.-A.’s FPU predoctoral contract is supported by the Spanish Ministerio de Educacion, Cultura y ́ Deporte. A.M.-C. is supported by a predoctoral contract of the Fundacion Tatiana Perez de Guzman el Bueno.

Many allergens feature hydrophobic cavities that allow the binding of primarily hydrophobic small‐molecule ligands. Ligand‐binding specificities can be strict or promiscuous. Serum albumins from mammals and birds can assume multiple conformations that facilitate the binding of a broad spectrum of compounds. Pollen and plant food allergens of the family 10 of pathogenesis‐related proteins bind a variety of small molecules such as glycosylated flavonoid derivatives, flavonoids, cytokinins, and steroids in vitro. However, their natural ligand binding was reported to be highly specific. Insect and mammalian lipocalins transport odorants, pheromones, catecholamines, and fatty acids with a similar level of specificity, while the food allergen β‐lactoglobulin from cow's milk is notably more promiscuous. Non‐specific lipid transfer proteins from pollen and plant foods bind a wide variety of lipids, from phospholipids to fatty acids, as well as sterols and prostaglandin B2, aided by the high plasticity and flexibility displayed by their lipid‐binding cavities. Ligands increase the stability of allergens to thermal and/or proteolytic degradation. They can also act as immunomodulatory agents that favor a Th2 polarization. In summary, ligand‐binding allergens expose the immune system to a variety of biologically active compounds whose impact on the sensitization process has not been well studied thus far.

The influence of gastrointestinal digestion on the immunological properties of three different nonspecific lipid-transfer proteins (nsLTPs) described in tomato fruit has been assessed using an

The influence of gastrointestinal digestion on the immunological properties of three different nonspecific lipid-transfer proteins (nsLTPs) described in tomato fruit has been assessed using an in vitro system mimicking the stomach and intestine digestion conditions. Tomato peel/pulp nsLTP, Sola l 3, was degraded after digestion, although the immunoglobulin E (IgE) recognition of intact protein and a 10 kDa band were still observed after 30 min of duodenal digestion in the presence of phosphatidylcholine. The tomato seed nsLTP, Sola l 7, showed a higher stability than the other seed allergen, Sola l 6, during digestion. Sola l 7 showed an IgE immunoreactive 6.5 kDa band in immunoblotting analysis, retaining up to 7% of its IgE-binding capacity in inhibition ELISA test after 60 min of duodenal digestion and keeping intact its ability to activate basophils after digestion. These results suggest that the tomato seed allergen Sola l 7 might be considered as an important allergen in the induction of allergic responses to tomato due to its high stability against gastrointestinal digestion., This work was supported by grants SAF2017-86483-R from the Ministerio de Economía y Competitividad and PI13/00928 Instituto de Salud Carlos III (ISCIII), ISCIII cofounded by Fondo Europeo de Desarrollo Regional – FEDER for the Thematic Networks and Co-operative Research Centres; RIRAAF RD16/0006/0024 and ARADyAL (RD16/0006/0001; RD16/0006/0013; RD16/0006/0014); and EAACI Exchange Research Fellowship 2016. The authors would like to thank the excellent technical support of Sara Abián. S.B. was supported by a Juan de la Cierva Incorporación contract from the Spanish Ministerio de Ciencia, Innovación y Universidades.

Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specific-immunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgE-binding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed.

This paper describes the first biosensor reported to date for the determination of mustard seed traces. The biosensor consists of an amperometric immunosensing platform able to sensitively and selectively determine Sin a 1 content, the major allergen of yellow mustard and the most abundant protein of these seeds. The immunosensing platform exploits the coupling of magnetic microbeads (MBs) modified with sandwich-type immune complexes, comprising polyclonal and monoclonal antibodies, selective to the target protein for its capturing and detection, respectively. In addition, a HRP-conjugated secondary antibody was used for enzymatic labelling of the monoclonal antibody, and amperometric transduction was made at screen-printed carbon electrodes (SPCEs) using the hydroquinone (HQ)/H2O2 system. The electrochemical immunosensor allows the simple and fast detection (a single 1-h incubation step) of Sin a 1 with a limit of detection of 0.82 ng mL−1 (20.5 pg of protein in 25 μL of sample) with high selectivity against structurally similar non-target allergenic proteins (such as Pin p 1 from pine nut). The developed immunoplatform was successfully used for the analysis of peanut, rapeseed, cashew, pine nut and yellow mustard extracts, giving only positive response for the yellow mustard extract with a Sin a 1 content, in full agreement with that provided by conventional ELISA methodology.

5 Pág. Departamento de Tecnología de Alimentos​​, The epitopes of the major allergen of pine nut, Pin p 1, were analyzed using a peptide library and sera from patients with clinical allergy to pine nut in order to deepen into the allergenic characteristics of Pin p 1. Analyses of epitope similarities and epitopes location in a 3D-model were also performed. Results showed that three main regions of Pin p 1 containing 5 epitopes were recognized by patient sera IgE. The epitopes of Pin p 1 had important similarities with epitopes of allergenic 2S albumins from peanut (Ara h 2 and 6) and Brazil nut (Ber e 1). The epitopes of Pin p 1 were found in α-helices and coils in the 3D protein structure. Interestingly, all epitopes were found to be well-exposed in the protein surface, which suggests facile access for IgE-binding to the structure of Pin p 1 which is known to be highly resistant., This work was supported by a grant from the research program Talento Investigador of the Community of Madrid (Regional Ministry of Science, Universities, and Innovation, Madrid, Spain), and a grant from Strategic Health Action (AES 2020), Carlos III Health Institute, Spanish Ministry of Science and Innovation (to B.C) (Grant number: PI20/00351). B.C. is senior researcher in the research program Talento Investigador of the Community of Madrid (number: 2019-T1/BIO-12690). The funding sources had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the article for publication.

This paper describes the first immunosensor reported so far for the highly sensitive determination of the egg white allergen, ovomucoid (OM). The approach involves the selective capture of sandwich immunocomplexes formed by capture antibody-target protein allergen-HRP-labeled detector antibody onto carboxylic acid-functionalized magnetic beads (HOOC-MBs). The resulting magnetic bioconjugates were captured on the surface of disposable screen-printed carbon electrodes (SPCE) to perform amperometric detection at −0.20 V vs. the Ag pseudo-reference electrode in the presence of hydroquinone (HQ) and H2O2. The as prepared electrochemical immunoplatform showed a wide range of linearity (0.3–25 ng mL−1), a LOD of 0.1 ng mL−1, which is much lower than those claimed for commercial ELISA kits, and a great selectivity against other white egg allergenic proteins. The developed immunosensor was successfully employed for the simple and accurate determination of trace amounts of OM in unprocessed (eggs and wheat flour) and baked (bread) food samples.

Airway epithelium is the cellular structure with the greatest surface exposed to a plethora of environmental airborne substances, including microorganisms, respiratory viruses, air pollutants, and allergens. In addition to being a protective physical barrier at the air-liquid interface, the airway epithelium acts as an effective chemical and immunological barrier that plays a crucial role in orchestrating the immune response in the lungs, by supporting the activation, recruitment, and mobilization of immune cells. Airway epithelium dysfunction has been clearly associated with various airway inflammatory diseases, such as allergic asthma. Although it is not fully understood why a person develops respiratory allergy, a growing body of evidence shows that the nature of the host's immune response is strongly determined by the state of the airway epithelium at the time of contact with the inhaled allergen. Our review highlights the physiological state of airway epithelium as a key element in the development of allergy and, particularly, in exacerbation of asthma. We review the role of physiological oxidants as signaling molecules in lung biology and allergic diseases and examine how high exposure to air pollutants (eg, cigarette smoke and diesel particles) can contribute to the increased incidence of respiratory allergy and exacerbation of the disease.

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.

Background Ole e 7 is a nonspecific lipid transfer protein (nsLTP) from olive pollen, one of the main allergenic pollens worldwide. This allergenic nsLTP is responsible for severe symptoms in regions with high olive pollen exposure, where many Ole e 7-sensitized patients exhibit a co-sensitization to the peach nsLTP, Pru p 3. However, there is no evidence of cross-reactivity, which explains this observed co-sensitization. Therefore, the purpose of this study was to explore the relationship between Ole e 7 and Pru p 3. Methods A total of 48 patients sensitized to Ole e 7 and/or Pru p 3 were included in the study. Specific IgE serum levels were measured by ImmunoCAP 250 and ELISA. Inhibition assays were performed to determine the existence of cross-reactivity between both nsLTPs. Allergic response was analyzed ex vivo (basophil activation test) and in vitro (RBL-2H3 mast cell model). Results Common IgG and IgE epitopes were identified between both allergens. IgE-binding inhibition was detected in Ole e 7-monosensitized patients using rPru p 3 as inhibitor, reaching inhibition values of 25 and 100%. Ex vivo and in vitro assays revealed a response against rPru p 3 in four (31%) Ole e 7-monosensitized patients. Conclusions Our results suggest that Ole e 7 could play a new role as primary sensitizer in regions with high olive pollen exposure, leading to the peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.

A novel and disposable electrochemical biosensor for PCR-free and selective detection of Sola l 7, a non-specific lipid transfer protein (nsLTP) found in tomato seeds associated to severe symptoms of tomato-allergic patients, is reported in this work. The methodology involves the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sola l 7 allergen coding sequence with appropriate RNA probes designed and described for the first time in this work. Labeling was carried out with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with HRP onto the surface of magnetic beads. Amperometric transduction was performed upon magnetic capture of the resulting magnetic bioconjugates on screen-printed electrodes using the system H2O2/HQ. A comparison of the sandwich hybridization format with a direct approach as well as between different labeling strategies was performed. The LOD value achieved was 0.2 pM (5 amol in 25 μL). The biosensor was successfully applied to the selective analysis of the targeted Sola l 7 specific region directly in just 100 ng of non-fragmented denatured genomic DNA extracted from tomato seeds., The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2015-64402-C2-1-R, SAF2017-86483-R and the TRANSNANOAVANSENS Program from the Comunidad de Madrid (Grant P2018/NMT-4349) are gratefully acknowledged. M.F.B. is grateful to FCT grant SFRH/BPD/78845/2011,financed by POPH–QREN–Tipologia4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologiae Ensino Superior. The author M.A.P.B. is grateful to the authors J.M.R., M.F.B., S.C., J.M.P. for the scientific assistance and suggestions shared throughout the supervision of her Master’s project and Master’s thesis at University of Minho. The author M.A.P.B. also acknowledges the Department of Biology (DB) and the Centre of Molecular and Environmental Biology (CBMA) from University of Minho (UM), Braga,Portugal, by providing all the conditions leading to the Master's thesis in“Biophysics and Bionanosystems”. Financial support of M.A.P.B. forthis work was provided by a fellowship within the programme Erasmus+ scholarship/Portugal, which allowed performing the laboratorial work at the University Complutense of Madrid, Madrid, Spain. The author J.M.R. acknowledges CBMA-UM and DB-UM, Portugal, by the conditions provided. J.M.R. is also grateful to the Green Chemistry Laboratory (LAQV)–Research center Chemistry and Technology Network (REQUIMTE), and the Department of Chemistry and Biochemistry (DCB) from Faculty of Sciences, University of Porto(FCUP), Porto, Portugal, where currently he is researcher. J.M.R. acknowledges the financial support of the strategic programmes UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and PTDC/SAUNUT/30448/2017 (POCI-01-0145-FEDER-030448) and M.F.B. the financial support of the projects PTDC/QUI-QAN/30735/2017 and UID/QUI/50006/2019, funded by national funds through Fundação para a Ciência e Tecnologia [Foundation for Science and Technology](FCT-I.P.)/Ministério da Ciência, Tecnologia e Ensino Superior [Ministry of Science, Technology and Higher Education] (MCTES), and Fundo Europeu de Desenvolvimento Regional [European RegionalDevelopment Fund] (FEDER), under the scope of the COMPETE2020–Programa Operacional Competitividade e Internacionalização[COMPETE2020–Competitiveness and InternationalizationOperational Program, POCI]. S.B. acknowledges the financial support from MINECO through the“Juan de la Cierva” program.

Olive pollen is a major cause of immunoglobulin E (IgE)-mediated allergy in Mediterranean countries. It is expected to become a worldwide leading allergenic source because olive cultivation is increasing in many countries. Ole e 15 belongs to the cyclophilin pan-allergen family, which includes highly cross-reactive allergens from non-related plant, animal and mold species. Here, the amino acid differences between Ole e 15 and its weak cross-reactive human homolog PPIA were grafted onto Ole e 15 to assess the contribution of specific surface areas to the IgE-binding. Eight Ole e 15-PPIA chimeras were produced in E. coli, purified and tested with 20 sera from Ole e 15-sensitized patients with olive pollen allergy by ELISA experiments. The contribution of linear epitopes was analyzed using twelve overlapping peptides spanning the entire Ole e 15 sequence. All the patients displayed a diverse reduction of the IgE-reactivity to the chimeras, revealing a highly polyclonal and patient-specific response to Ole e 15. IgE-epitopes are distributed across the entire Ole e 15 surface. Two main surface areas containing relevant conformational epitopes have been characterized. This is the first study to identify important IgE-binding regions on the surface of an allergenic cyclophilin. We thank the excellent technical support of Sara Abián Saz. This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional (FEDER): SAF2014-53209-R to M.V. and R.B. and SAF2017-86483-R to M.V. from the Ministerio de Economía y Competitividad and by the Thematic Networks and Co-operative Research Centres: RIRAAF Network RD12/0013/0015; and ARADyAL (RD16/0006/0014) from the Instituto de Salud Carlos III (ISCIII). A.N. and A.J. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the Alergosur Foundation. R.B. also acknowledges the financial support of the PI17CIII/00045 grant from the AES-ISCIII program. The FPU predoctoral contract to P.S.S.-A. is supported by the Spanish Ministerio de Educación, Cultura y Deporte. C.O.-S. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. Sí

Allium genus plants, such as leek (Allium porrum), are rich sources of anti-inflammatory and anti-oxidant secondary metabolites, this is of interest because it demonstrates their suitability as pharmacological alternatives for inflammatory processes, including allergy treatment. The composition of methanolic leek extract (LE) was analyzed by GC&ndash, MS and LC&ndash, IT/MS, and the total phenolic content and antioxidant capacity were quantified by colorimetric methods. Its pharmacological potential was analyzed in human bronchial epithelial Calu-3 cells, human mast cells LAD2, and humanized rat basophiles RBL-2H3. LE exhibited a cytotoxic effect on Calu-3 cells and HumRBL-2H3 cells only at high concentrations and in a dose-dependent manner. Moreover, LE decreased the degranulation of LAD2 and HumRBL-2H3 cells. LE treatment also significantly prevented alterations in transepithelial electrical resistance values and mRNA levels of glutathione-S-transferase (GST), c-Jun, and NF&kappa, B after treatment with H2O2 in ALI-cultured Calu-3 cells. Finally, ALI-cultured Calu-3 cells treated with LE showed lower permeability to Ole e 1 compared to untreated cells. A reduction in IL-6 secretion in ALI-cultured Calu-3 cells treated with LE was also observed. In summary, the results obtained in this work suggest that A. porrum extract may have potential anti-allergic effects due to its antioxidant and anti-inflammatory properties. This study provides several important insights into how LE can protect against allergy.

2S albumins are relevant and often major allergens from several tree nuts and seeds, affecting mainly children and young people. The present study aims to assess how the structural features of 2S albumins could affect their immunogenic capacity, which is essential to comprehend the role of these proteins in food allergy. For this purpose, twelve 2S albumins were isolated from their respective extracts by chromatographic methods and identified by MALDI-TOF mass-spectrometry. Their molecular and structural characterization was conducted by electrophoretic, spectroscopic and in silico methods, showing that these are small proteins that comprise a wide range of isoelectric points, displaying a general high structure stability to thermal treatment. Despite low amino acid sequence identity, these proteins share structural features, pointing conformational epitopes to explain cross-reactivity between them. Immunoblotting with allergic patients’ sera revealed those possible correlations between evolutionarily distant 2S albumins from different sources. The availability of a well-characterized panel of 2S albumins from plant-derived sources allowed establishing correlations between their structural features and their allergenic potential, including their role in cross-reactivity processes.

Aeroallergens are airborne substances-mainly proteins-capable of triggering Th2-immune responses in respiratory allergies. They enter into the body through the upper airways, reaching the mucosa afterward. Mucosae lining at the luminal side consists of an epithelial barrier completely covered by mucus and pulmonary surfactant. Both pulmonary surfactant and plasma membrane of the epithelial cells represent two physiological phospholipid-based barriers where allergens first impact before triggering their biological effects. The interaction of allergens with lipids at relevant physiological surfaces could promote structural changes on the molecule, resulting on a potential modification of its allergenic properties. In this work, we have first described the surface and phospholipid interaction capabilities of the clinically relevant aeroallergen Ole e 1, the main allergen of olive tree pollen. By using epifluorescence microscopy of Langmuir transferred films, we observed that lipid-packed ordered domains may function as a preferential location for allergen to accumulate at the air-liquid interface, an effect that is abolished in the presence of cholestenone. The possible implications of phospholipid-interfacial effects in the modification of allergen structural and functional properties will be discussed.

Background Ingestion of food allergens present in maternal milk during breastfeeding has been hypothesized as a gateway to sensitization to food; however, this process could develop during pregnancy, as the maternal–fetal interface develops a Th2- and Treg-mediated environment to protect the fetus. We hypothesized that in these surroundings, unborn children are exposed to food allergens contained in the mother's diet, possibly giving rise to first sensitization. Methods The presence of allergens in utero was studied by analyzing amniotic fluid (AF) samples in two different stages of pregnancy: at 15–20 weeks and after delivery at term. An antibody microarray was developed to test for the most common food allergens. The array detects the presence of ten allergens from milk, fruit, egg, fish, nuts, and wheat. Results AF from 20 pregnant women was collected: eight after delivery at term and 12 from women who underwent diagnostic amniocentesis between weeks 15 and 20 of gestation. The presence of allergens was detected in all samples. Samples from amniocentesis had a higher allergen concentration than samples after delivery at term. Conclusions We demonstrated the presence of intact major food allergens in AF samples. This early contact could explain subsequent sensitization to foods never eaten before.

Although pizza is one of the most popular foods in the world, allergic responses after ingesting pizza are relatively uncommon. However, precisely identifying the allergens responsible for these allergic reactions is challenging because of the high and diverse number of ingredients used in pizza preparation. In this report, we aim to identify the allergens responsible for systemic allergic reactions following ingestion of pizza in two patients. Using a skin prick by prick test (SPPT) and in vitro techniques, with natural and recombinant purified allergens from tomato and mustard seeds, we identified 2S albumin and non-specific lipid transfer proteins (nsLTP) as the proteins involved. However, IgE responses to the four nsLTPs differed before and after denaturation and reduction, thus suggesting additional complexity around nsLTP in food processing.

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways. This work was supported by SAF2014-53209-R and SAF2017-86483-R grants from the Ministerio de Economía y Competitividadto R.B. and M.V., and to M.V., respectively, RTI2018-094564-B-I00 from the Spanish Ministry of Science and Universities andP2018/NMT4389 from the Regional Government of Madrid to J.P-G. and A.C., and RIRAAF Network RD12/0013/0015 grant and ARADyAL Network RD16/0006 grant from the Instituto de Salud Carlos III (ISCIII) co-founded by Fondo Europeo de Desarrollo Regional -FEDER-for the Thematic Networks and Co-operative Research Centres.A.J. and C.M. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the Alergosur Foundation. JC.L-R., P.SS-A. and C.G-M. are recipients of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. We also gratefully acknowledge the financial support from theAES-ISCIII for the PI17CIII/00045 grant to R.B. We also thank the excellent technical support of Sara Abián. Sí

[This corrects the article DOI: 10.1371/journal.pone.0230010.].

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.

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.