1020
Specificity of non specific lipid transfer proteins and influence of the ligands on their three dimensional structure
Dubiela, P1; Humeniuk, P1; Pfeifer, S1; Bublin, M1; Cantini, F2; Borowski, T3; Alessandri, S4; Hoffmann-Sommergruber, K1
1Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria;2Center for Magnetic Resonance, University of Florence, Florence, Italy;3The Jerzy Haber Institute of Catalysis and Surface Chemistry of the Polish Academy of Sciences, Krakow, Poland;4Department of Statistics, Computer Science, University of Florence, Applications
‘G. Parenti’ (DISIA), Florence, Italy
Background: Plant non-specific lipid trans-fer proteins (nsLTPs) are relevant food allergens e.g. from peach (Pru p 3), hazel-nut (Cor a 8) or walhazel-nut (Jug r 3). They share a conserved fold with an internal cavity. Different lipid-protein complexes showed that the tunnel adapts its volume while binding a broad range of hydropho-bic molecules.
Method: The binding of lipids to Pru p 3, Cor a 8, and Jug r 3 was monitored by adding 10lM 1,8-ANS and measuring the decrease of 1,8-ANS fluorescence. Further-more, molecular dynamic analysis (MD) was applied to explore the nature of inter-action between nsLTPs and tested ligands.
Saturation transfer difference (STD) spec-troscopy and W-LOGSY (Water-Ligand Observed via Gradient Spectroscopy) tech-nique were applied to confirm results obtainedin silico.
Results: Due to pre-incubation of Pru p 3 with lipids a concentration dependent reduction of ANS binding was observed.
Pru p 3 incubated (1:1; 1:10) with lauric acid showed 19% and 66% of ANS fluo-rescence reduction respectively, compared with Pru p 3 without lipids. For oleic acid (1:1; 1:10) reduction was 7% and 77%, respectively. Molecular dynamic analysis suggests changes in protein structure due to binding to certain ligands. Interaction between oleic acid and Pru p 3, moved the C-terminal loop out towards the surface of the molecule. NMR based experiments confirmed binding capacity observed in MD analysis.
Conclusion: In this study, we observed dif-ferences in binding capacity of Pru p 3.
Molecular dynamic simulation has shown that interaction between Pru p 3 and tested ligands can lead to conformational
changes. The allergen-lipid interaction may act as a potential danger signal during the allergic sensitization phase or increase allergenicity during the effector phase.
1021
Molecular, structural and immunological characterization of Der p 18, a chitinase-like house dust mite allergen
Resch, Y1; Blatt, K2; Malkus, U3; Fercher, C4; Swoboda, I1,5; Focke, M1; Chen, K-W1; Seiberler, S1; Mittermann, I1; Lupinek, C1; Rodriguez-Dominguez, A1; Zieglmayer, P6; Zieglmayer, R6; Keller, W4;
Krzyzanek, V7; Valent, P2; Valenta, R1; Vrtala, S1
1Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria;
2Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria;3Institute of Medical Physics and Biophysics, University of Munster, M€ unster,€ Germany;4Institute of Molecular Biosciences, University of Graz, Graz, Austria;5Section of Molecular Biotechnology, University of Applied Science, Vienna, Austria;6Vienna Challenge Chamber, Vienna, Austria;
7Academy of Sciences of the Czech Republic, Institute of Scientific Instruments of the ASCR, Czech Republic
Background: The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.
Objective: To perform a detailed charac-terization of Der p 18 on a molecular, structural and immunological level.
Methods: Der p 18 was expressed in E. coli, purified to homogeneity and its sec-ondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold elec-tron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects).
IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n=98) and its allergenic activity was analyzed in basophil activation experiments.
Results: Recombinant Der p 18 was expressed and purified as a folded protein.
Despite considerable sequence identities with Der f 18 fromDermatophagoides fari-nae and proteins from the other tested allergen sources, no relevant cross-reactiv-ities were found. The allergen was mainly localized in the peritrophic matrix of the
HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensi-tized patients.
Conclusion: Der p 18 seems to be a spe-cies-specific minor allergen but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.
1022
Amb a 1 isoforms show distinct IgE-binding properties
Wolf, M1; Twaroch, T2; Hauser, M1; Ebner, C3; Briza, P1; Steiner, M1; Aglas, L1; Behrendt, H4; Neubauer, A2; Stolz, F2; Ferreira, F1; Wallner, M1
1Department of Molecular Biology, University of Salzburg, Salzburg, Austria;2Biomay AG, Vienna, Austria;3Allergieambulatorium am Reumanplatz, Vienna, Austria;4ZAUM, Center for Allergy and Environment, Munich, Germany
Background: Ragweed pollen allergies rep-resent a serious health problem throughout vast regions of North America as well as Europe, and are an emerging risk factor for respiratory allergies in Asia. The major allergen of short ragweed, Amb a 1, has been identified as member of the pectate lyase allergen family. To date five different Amb a 1 isoforms have been acknowledged by the WHO/IUIS allergen nomenclature subcommittee.
Methods: Thus, we performed mass spec-trometry analyses of aqueous ragweed pol-len extracts to quantify the isoform composition of Amb a 1. Based on our data, three highly abundant Amb a 1 iso-forms were purified from natural extracts.
Moreover, one oft these isoforms was pro-duced as recombinant protein in P. pastoris. The allergens were fully char-acterized and their IgE-binding properties were compared by immunoblot, ELISA and mediator release assays.
Results: Amb a 1 isoforms show a consid-erable degree of sequence heterogeneity.
The predicted structure of Amb a 1 is char-acterized by a central parallel-beta-helix surrounded by alpha helical fold motives.
Our investigations of secondary structure elements by CD and FTIR spectroscopy revealed slight differences between the
Amb a 1 isoforms. This translated into dif-ferent IgE-binding properties of Amb a 1 isoforms in mediator release assays.
Conclusion: Analyses of pollen extracts demonstrated that several Amb a 1 iso-forms are present in rather similar quanti-ties in ragweed pollen. Therefore, the differences of IgE-binding to Amb a 1 iso-forms could be a result of different sensi-tizing properties. This should be taken into consideration when selecting particular rag-weed pollen isoforms for allergy diagnosis as well as therapy.
Acknowledgments: This work was sup-ported by FWF project L688, CK-CARE AG Individual Project 2009-02, Biomay AG, the Christian Doppler Research Association, and Sparkling Science SPA 05-193.
1024
Update on AllergenOnline.org: a comprehensive, searchable database for risk assessment
Goodman, RE1; Baumert, JL1; Taylor, SL1; Lalithambika, S1; Sampson, HA2; Ebisawa, M3; Ferriera, F4; Bohle, B5; van Ree, R6
1Food Allergy Research and Resource Program, University of Nebraska, Lincoln, NE, United States;
2Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY, United States;
3Departmeng of Allergy, Sagamihara National Hospital, Sagamihara, Japan, ;4Department of Molecular Biology, University of Salzburg, Salzburg, Austria;
5Department of Pathophysiology, University of Vienna, Vienna, Austria;6Departments of Experimental Immunology and Otorhinolaryngology, Academic Medical Center, Amsterdam, the Netherlands
Background: Food safety regulators in many countries are demanding evaluation of the potential allergenicity of genetically engineered food crops and processed foods containing novel proteins. Currently no single or combinations of laboratory tests are highly predictive at gauging the poten-tial allergenicity of individual proteins.
However, practical clinical experience has demonstrated the common, serious risk of food allergy (anaphylaxis) are due to the unexpected consumption of proteins from known allergenic sources by consumers who have been diagnosed and told to avoid their allergens. In some cases cross-reactive IgE binding to homologous pro-teins causes severe reactions. The Aller-genOnline database, established in 2005 is updated annually and curated by our expert panel to provide a sequence-search-able bioinformatics tool to identify poten-tially risky proteins before they are transferred to new food sources.
Method: Each year the NCBI Protein database and the WHO/IUIS Allergen database are searched for newly identified candidate allergens. Published scientific literature is searched using PubMed for
data demonstrating protein-specific IgE binding from donors allergic to the source of the protein. Evidence of IgE binding to cross-reactive carbohydrate determinants is evaluated along with characterization of the protein (expression in the source mate-rial and amino acid sequence or expected translation product). Additionally evidence of biological activity related to specific IgE binding is sought (basophil activation, skin prick tests or challenges) to support the designation as an allergen. The amino acid sequences are compiled and relevant refer-ences are listed for each taxonomic protein group.
Results: Version 16 will be released 26 Jan-uary, 2016. It includes 1956 sequences from 778 taxonomic protein groups. Version 15 included 1897 proteins in 744 groups. Use of the database is free to the public. We do not collect user or query sequence informa-tion. Users enter the amino acid sequence of their protein of interest to search for align-ments with identities that might conserva-tively predict potential cross-reactivity as described online with alignment identity and Escores.
Conclusion: Search results should guide developers and regulators to accept the protein as unlikely to present a known risk of immediate allergic elicitation or alterna-tively identifies the protein as an allergen or one that should require specific serum tests.
1025
Bet v 1-homologous proteins from chickpea show IgE cross reactivity in patients with birch pollen allergy
Kulkarni, A1; Wangorsch, A2; Blanca-Lopez, N3; Ferrer, M4; Ananthanarayan, L1; Vieths, S2; Toda, M2; Scheurer, S2
1Food Engineering & Technology Department, Institute of Chemical Technology, Mumbai, India;2 Paul-Ehrlich-Institut, Division of Allergology, Langen, Germany;
3Infanta Leonor Hospital, Allergy Service, Madrid, Spain;4Department of Allergy, School of Medicine, Pamplona, Spain
Background: Homologues of Bet v 1, the major birch pollen allergen, from legume foods are mainly involved in allergic reactions caused by IgE cross-reactivity.
Chickpeas have been reported to cause IgE-mediated hypersensitivity reactions in patients from Spain & India, particularly in pediatric patients. So far no chickpea allergens are listed in the official IUIS allergen database. The objective of the pre-sent study was the cDNA cloning, recom-binant expression and immunological characterization of Bet v 1 homologous PR10-proteins from chickpea.
Method: Two Bet v 1 homologous PR10 proteins from chickpea which were
predicted by an in silico analysis to be potential allergens. The pre synthesized cDNA sequences, designated as Cic a 1.01 (Q9SMK8) and Cic a 1.02 (Q39450), were expressed in E. coli. Cic a 1.01 was purified under native conditions by DEAE & size exclusion chromatography (SEC) whereas Cic a 1.02 was purified under denaturing conditions by MonoQ & SEC. Purity &
secondary structure of the proteins were analyzed by SDS-PAGE, CD spectroscopy, DLS and LC-MS. Serum samples of 9 birch allergic patients who were sensitized to Bet v 1 along with 26 sera samples of chickpea allergic patients from Spain were investigated for IgE reactivity with raw &
boiled chickpea extract, purified Cic a 1.01
& Cic a 1.02.
Results: Cic a 1.01 (AJ275304) & Cic a 1.02 (X79706) showed 64% amino acid identity (aa-id) to one another and 49%
and 44% aa-id to Bet v 1 (X15877), respec-tively. Both recombinant proteins displayed secondary structures similar to those of Bet v 1. IgE detection of raw chickpea extract with Spanish chickpea allergic patient’s serum samples resulted in multiple bands.
2 out of 26 Spanish sera showed IgE reac-tivity with these proteins. IgE detection of raw & boiled extract with Bet v 1 cross reactive sera resulted in multiple bands and few of these sera also showed reactivity with purified proteins. IgE binding with raw chickpea extract was inhibited at a sin-gle band using recombinant purified Bet v 1 homologous chickpea proteins, confirm-ing the presence of these proteins in chick-pea.
Conclusion: Chickpea allergy might be mediated by means of cross-reaction with the Bet v 1 homologue from chickpea in a subgroup of patients allergic to Birch, Bet v 1. These proteins were not seen to be so relevant for Spanish chickpea allergic patients. The allergenic potency for these proteins needs to be further evaluated to determine its clinical relevance.
1026
Supporting diagnostic test allergens used forin vivodiagnosis of allergic disease: a case study
Heath, MD; Starchenka, S; Mwange, J; Swan, N;
Hewings, S; Skinner, MA
Allergy Therapeutics plc., Worthing, United Kingdom
Background: It remains of paramount importance for the clinician to have avail-able a comprehensive range of high-quality diagnostic tests for in vivo diagnosis of IgE-mediated allergies. However, the regu-latory demands and associated costs for manufacturers in maintaining a compre-hensive ‘gold-standard’ skin-prick test
portfolio has become increasingly challeng-ing, and as such, is driving manufacturers to significantly limit their test allergen (TA) portfolio. In a recent review of the situation, Klimek and colleagues, 2015, presented a number of regulatory consider-ations that might help facilitate the mainte-nance of TAs in Europe. Of note, was to promote the concept of homologous groups. The work presented herein pro-vides a route to assess inclusion criteria for new Tree and Epithelial allergens within their respective homologous groups, in line with EMA guidance, providing scope for manufacturers to extrapolate quality data where appropriate, thus facilitating TA maintenance.
Methods: Structural homology was assessed for epithelial allergens (Lipocalins) from cat, dog, guinea pig, hamster and horse. In addition to this, their biological cross-reactivity was compared using diag-nostic formulations in separate ELISA experiments using cat IgG rabbit sera and IgE human sera.
Identical assessments for Plane and Lime Tree allergens with the ‘Birch’ group were also made. The comparability of biochemi-cal stability of different allergen sources was assessed through statistical methods for the aforementioned tree and epithelial species.
Results: Allergen cross-reactivity and/or structural homology have been described providing justification for inclusion of Epithelial and Tree allergens within homol-ogous group formations. Statistical physic-ochemical/biological similarities among mammalian epithelial species provided for further justification in accordance with EMA guidance. An example of how stabil-ity data of TAs may be extrapolated within the same homologous group was further assessed.
Conclusions: The concept of homologous groups is dynamic allowing the flexibility and potential in streamlining quality parameters, such as stability profiles, due to extrapolation of exemplar data to a wider range of allergens. The work pre-sented herein pays tribute to the concept of homologous groups providing an assess-ment of the current literature and support-ing data for establishsupport-ing a homologous group for Epithelials and extending the
‘Birch’ tree group.
1027
First case series: exotic pets allergy among Chilean patients
Tordecilla, R1,2; Aguilera, R1; Marinovic, MA2; Bastıas, C1; Salinas, J3
1Clinical Hospital University of Chile, Santiago, Chile;
2Santa Marıa Clinic, Santiago, Chile;3Las Condes Clinic, Santiago, Chile
Background: The most frequent pet allergy is allergy to cats and dogs, but exposure to other nontraditional pets, such as rodents (eg, mice, hamsters, guinea pigs), other mammals (eg, ferrets, hedgehogs, pigs, monkeys), spiders (eg, tarantulas), reptiles (eg, snakes), and exotic birds has increased in recent years inducing new allergic sensi-tization, frequently manifested by respira-tory or cutaneous symptoms.
Objective: To characterize the first cases series of chilean patients with exotic pet al-lergy.
Results: In this report we describe five patients allergic to exotic pets, four women and one man (median age 30 years old). Pets included three hedge-hogs, one ferret and one guinea pig. The clinical symptoms included rhinoconjunc-tivitis, contact dermatitis, urticaria, angioedema and bronchial obstruction.
All patients were tested with common aeroallergens, a battery of animal epithe-lium commercial extracts (cat and dog), and extracts prepared with dander and spine (hedgehogs) or dander and hair (fer-ret and guinea pig) (unavailability of com-mercial extract). All patients allergic to hedgehog showed allergic rhinoconjunc-tivitis and two of them also had bronchial hyperreactivity. All of them had positive skin prick test (SPT) to spines and dander extract. One of these patients tested posi-tive also to SPT with dust mite. The only patient with contact dermatitis owned a ferret, and had negative SPT to his pet, but a positive patch test at 72 h to the dander and hair ferret extract. One patient presented severe persistent rhinoconjunctivitis with the exposure to guinea pig, she had positive IgE levels for guinea pig (0.7 UI/ml) and tested positive to SPT with dust mite and grass polen commercial extracts. Only two patients of this report were atopic and had other sen-sibilizations. All patients improved their symptoms when their pets were removed or not had direct contact with them, except for one patient who showed no response despite she was sensitized only to her pet (hedgehog) and not to any other inhalant.
Conclusion: The allergy to exotic pets like hedgehog, ferret and guinea pig occur in our country, so it is necessary to be aware that some symptoms may not be caused by common inhalants. It is important to study
this allergy, so recommendations like pet removal can help to improve symptoms.
1028
Hedgehog allergy
Haroun-Dı´az, E1; Vazquez de la Torre Gaspar, M1; Garcimartın Galicia, MI1; Ruano Perez, F1; Perez Alzate, D1; SomozaAlvarez, ML 1; Blanca Lopez, N1; Pastor Vargas, C2; Bartolome, B3; Canto Dıez, G1
1Hospital Infanta Leonor, Madrid, Spain;2Fundacion Jimenez Dıaz, Madrid, Spain;3Departamento I+D, Bilbao, Spain
Background: Hedgehogs are any of the spiny mammals of the subfamily Erina-ceinae whose most distinctive trait are his quills, not poisonous sharp hollow hairs made with keratin that are used as a defense. When threatened, a hedgehog is able to curl up into a ball with its quills extended.
The long-eared hedgehog, known as African pygmy, is the most common type sold as pets.
Although two cases of adverse reactions after exposure to hedgehog have been reported, no cases of IgE-mediated allergy to hedgehog have been described in the literature.
The aim of this study was to present a case of IgE-mediated allergy and to iden-tify two relevant allergens.
A 23-year-old woman with a previous diagnosis of seasonal rhino-conjunctivitis presented with shortness of breath, ocular and nasal itching, runny nose and contact urticaria after exposure to hedgehog. She had an African pygmy hedgehog at home as a pet, and presented perennial symp-toms coinciding with the introduction of the animal at home.
Method: Skin prick tests (SPT) was per-formed with the most common aeroaller-gens in our environment.
Hedgehog extract was performed with small, round balls of hedgehog feces with urine obtained from the bedding material.
Protein bands were separated by SDS-PAGE and immunoblotting was performed to detect IgE binding bands from hedgehog extract. IgE binding bands were identified by mass spectrometry using liquid chromatography-tandem mass spectrometry.
Results: SPT was positive (wheal≥3 mm) to pollen - Phleum pratense, Olea euro-paea, Platanus acerifolia, Cupressus arizon-ica -, cat and dog epithelium and moulds (Alternaria, Aspegillus fumigatus and Cla-dosporium).
SDS-PAGE revealed bands between 97 and 9 kDa. The immunoblotting showed IgE- binding bands of 42, 40, 32 and 29 kDa identified by mass-spectrometry as acidic mammalian chitinase-like for the
40 kDa, carboxypeptidase A1 for the 32 kDa and chymotrypsin-like elastase family member (CELA) for the 29 kDa.
The 42 kDa IgE-binding band was not visualized in the SDS-PAGE and, thus, could not be analyzed.
Conclusion: We present a case of IgE-mediated to Hedgehog and we identify chitinase-like, carboxypeptidase A1 and chymotrypsin-like elastase as relevant allergens.
1029
Allergy to Marijuana: a case report
Alcantara Villar, M1; Can˜ada Pen˜a, CL1; Jimeno Nogales, L2; Castro de las Cuevas, L2; De La Torre
Alcantara Villar, M1; Can˜ada Pen˜a, CL1; Jimeno Nogales, L2; Castro de las Cuevas, L2; De La Torre