Studying the molecular and immunological basis of allergic diseases often requires purified native allergens. The methodologies for protein purification are usually difficult and may not be completely successful. The objective of this work was to describe a methodology to purify allergens from their natural source, while maintaining their native form. The purification strategy consists of a three-step protocol and was used for purifying five specific allergens, Ole e 1, Amb a 1, Alt a 1, Bet v 1 and Cup a 1. Total proteins were extracted in PBS (pH 7.2). Then, the target allergens were pre-purified and enriched by salting-out using increasing concentrations of ammonium sulfate. The allergens were further purified by anion exchange chromatography. Purification of Amb a 1 required an extra step of cation exchange chromatography. The detection of the allergens in the fractions obtained were screened by SDS-PAGE, and Western blot when needed. Further characterization of purified Amb a 1 was performed by mass spectrometry. Ole e 1, Alt a 1, Bet v 1 and Cup a 1 were obtained at > 90 % purity. Amb a 1 was obtained at > 85 % purity. Overall, we propose an easy-to-perform purification approach that allows obtaining highly pure allergens. Since it does not involve neither chaotropic nor organic reagents, we anticipate that the structural and biological functions of the purified molecule remain intact. This method provides a basis for native allergen purification that can be tailored according to specific needs.

2024-08-01·Allergy

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice

Article

作者: Jensen‐Jarolim, Erika ; Szepannek, Nathalie ; Pali‐Schöll, Isabella ; Berger, Markus ; Roth‐Walter, Franziska ; Fakhimahmadi, Aila ; Bianchini, Rodolfo ; Hofstetter, Gerlinde ; Hufnagl, Karin ; Wiederstein, Markus ; Jensen, Sebastian A.

AbstractBackgroundWe investigated the biological function of the mould allergen Alt a 1 as a carrier of micronutrients, such as the vitamin A metabolite retinoic acid (RA) and the influence of RA binding on its allergenicity in vitro and in vivo.MethodsAlt a 1‐RA complex formation was analyzed in silico and in vitro. PBMCs from Alternaria‐allergic donors were stimulated with Alt a 1 complexed with RA (holo‐Alt a 1) or empty apo‐Alt a 1 and analyzed for cytokine production and CD marker expression. Serum IgE‐binding and crosslinking assays to apo‐ and holo‐protein were correlated to B‐cell epitope analysis. Female BALB/c mice already sensitized to Alt a 1 were intranasally treated with apo‐Alt a 1, holo‐Alt a 1 or RA alone before measuring anaphylactic response, serum antibody levels, splenic cytokines and CD marker expression.ResultsIn silico docking calculations and in vitro assays showed that the extent of RA binding depended on the higher quaternary state of Alt a 1. Holo‐Alt a 1 loaded with RA reduced IL‐13 released from PBMCs and CD3+CD4+CRTh2 cells. Complexing Alt a 1 to RA masked its IgE B‐cell epitopes and reduced its IgE‐binding capacity. In a therapeutic mouse model of Alternaria allergy nasal application of holo‐Alt a 1, but not of apo‐Alt a 1, significantly impeded the anaphylactic response, impaired splenic antigen‐presenting cells and induced IL‐10 production.ConclusionHolo‐Alt a 1 binding to RA was able to alleviate Th2 immunity in vitro, modulate an ongoing Th2 response and prevent anaphylactic symptoms in vivo, presenting a novel option for improving allergen‐specific immunotherapy in Alternaria allergy.

2024-05-01·World Allergy Organization Journal

Bayesian analysis suggests independent development of sensitization to different fungal allergens

Article

作者: Mokin, Vitalii ; Shmundiak, Dmytro ; Kurchenko, Andrii ; Rodinkova, Victoria ; Yuriev, Serhii ; Kryzhanovskyi, Yevhenii ; Bortnyk, Mykyta ; Kryvopustova, Mariia

Fungi are known for their ability to cause allergies, but data on individual sensitization to them are insufficient.The purpose of the study was to carry out a comprehensive anal. of the fungal allergens′ sensitization profile in the Ukrainian population and to determine both population and individual sensitivity to these allergens.We utilized a set of ALEX allergy test data from 20,033 inhabitants of 17 regions of Ukraine from 1 to 89 years conducted in 2020-2022.A complex of programs in the Python language was developed and Bayesian network anal. was applied to determine the sensitivity combinations in individual patients to various fungal components.Sensitivity to Alt a 1 dominated and was observed in 79.39% of patients, and 62.17% of them were sensitive solely to Alt a 1.Exclusive sensitivity to Mala s 6 was second in individual patient profiles with a frequency of 4.06%.Combined sensitivity to Alt a 1 - Asp f 3 was third with a share of 3.28%.Pen ch and Cla h extracts stimulated the production of the lowest median sIgE levels.The highest median sIgE levels were for Alt a 1, Mala s 11 and Asp f 6, resp.Median sIgE levels increased in adults compared to children for all components of Aspergillus fumigatus, as well as for Mala s 5 and Mala s 11.In the rest of the cases, they decreased in adults compared to children.The sensitization rates to fungi in general and specifically to Alternaria were lower in the western parts of Ukraine, especially in the Carpathian region, situated within the Broad-leaved Forest zone.The results of Bayesian modeling revealed that in the case of Alt a 1, the simultaneous absence of sensitivity to Cla h 8, Mala s 11, Mala s 5 and Mala s 6 mols. could condition the presence of sensitization to the major Alternaria allergen with a probability of 92.42%.In all other cases, there was a high probability of absence of sensitivity to particular allergen against the background of absence of sensitivity to other ones, which may indicate the independent development of sensitization to different fungal allergens.Sensitivity to Alt a 1 dominated in the studied population with a lower rate in the western regions.The highest median sIgE levels were induced by Alt a 1, Mala s 11 and Asp f 6.Bayesian Anal. suggest a high probability of the independent development of sensitization to different fungal allergens.The idea that sensitization to one allergen may be protective against sensitization to another one(s) requires further clin. study.

100 项与 1-a 相关的药物交易

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