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BACKGROUND: Snail allergy is rare but can be fatal. Pila polita, a freshwater snail, was considered as a popular exotic food, particularly in tropical countries, and consumed in processed forms. Thus, the purpose of this study was to identify the major and cross‐reactive allergens of P. polita and to determine the impact of food processing on the allergen stability. RESULTS: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis fractionated raw snail extract to approximately 24 protein bands, between 9 and 245 kDa. The prominent band at 33 kDa was detected in all raw and processed snail extracts. Immunoblotting tests of the raw extract demonstrated 19 immunoglobulin E (IgE)‐binding proteins, and four of them, at 30, 35, 42 and 49 kDa, were revealed as the major IgE‐binding proteins of P. polita. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry identified the 49 and 42 kDa major allergens as actin, whereas the 30 and 35 kDa major allergens were identified as tropomyosin. Immunoblotting revealed that the raw snail had more allergenic proteins than the processed snail. The degree of allergenicity in decreasing order was raw > brine pickled> boiled > roasted > fried > vinegar pickled. The presence of cross‐reactivity between P. polita and the shellfish tested was exhibited with either no, complete, or partial inhibitions. CONCLUSION: Actin and tropomyosin were identified as the major and cross‐reactive allergens of P. polita among local patients with snail allergy. Those major allergens are highly stable to high temperatures, acidic pH, and high salt, which might played a crucial role in snail allergy in Malaysia. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Introduction: Cow epithelium allergy (CEA) has been described in workers highly exposed to cattle, such as farmers and veterinarians, being a health problem in this population since it is their main livelihood. This study aimed to characterize the main clinical manifestations and define the sensitization profile of the cow epithelium-allergic population treated in our health area. Methods: This is a retrospective study including a total of 34 patients with a clinical diagnosis of CEA, confirmed by skin tests, bovine epithelium-specific IgE levels and allergen-specific conjunctival challenge test in some cases. They were distributed by age, sex, profession, clinical symptoms, specific IgE levels to other mammalian epithelia, pollens, mites, and foods. Immunoblotting was performed with extracts from cow dander, cow body fluids (urine and saliva), bull urine, and 17 sera from immunotherapy-untreated CEA patients. Results: The mean age of the patients was 44 years, with a higher incidence in cattle farmers. Rhinoconjunctivitis occurred in 100% of cases, with 35% having monosensitization to cow epithelium. Sera from most patients detected a 20-kDa IgE-binding band in cow dander, cow saliva, cow urine, and bull urine, corresponding to the major allergen Bos d 2 (bovine lipocalin). In 70% of the patients, a 25-kDa band was detected in cow and bull urine extracts, whose identification by mass spectrometry and investigation with protein databases led to the identification of a Bos taurus lipocalin (UniProt protein ID: A0A3Q1LGU7_BOVIN). Conclusion: CEA should be considered in patients exposed to cattle and as a cause of occupational disease. The IgE immunodetection revealed sensitization to a protein present in cow and bull urine (odorant-binding protein) not previously described. [ABSTRACT FROM AUTHOR]

Termites live in colonies, and their members belong to different castes that each have their specific role within the termite society. In well-established colonies of higher termites, the only food the founding female, the queen, receives is saliva from workers; such queens can live for many years and produce up to 10,000 eggs per day. In higher termites, worker saliva must thus constitute a complete diet and therein resembles royal jelly produced by the hypopharyngeal glands of honeybee workers that serves as food for their queens; indeed, it might as well be called termite royal jelly. However, whereas the composition of honeybee royal jelly is well established, that of worker termite saliva in higher termites remains largely unknown. In lower termites, cellulose-digesting enzymes constitute the major proteins in worker saliva, but these enzymes are absent in higher termites. Others identified a partial protein sequence of the major saliva protein of a higher termite and identified it as a homolog of a cockroach allergen. Publicly available genome and transcriptome sequences from termites make it possible to study this protein in more detail. The gene coding the termite ortholog was duplicated, and the new paralog was preferentially expressed in the salivary gland. The amino acid sequence of the original allergen lacks the essential amino acids methionine, cysteine and tryptophan, but the salivary paralog incorporated these amino acids, thus allowing it to become more nutritionally balanced. The gene is found in both lower and higher termites, but it is in the latter that the salivary paralog gene got reamplified, facilitating an even higher expression of the allergen. This protein is not expressed in soldiers, and, like the major royal jelly proteins in honeybees, it is expressed in young but not old workers. [ABSTRACT FROM AUTHOR]

Bet v 1 is the major allergen in birch pollen to which up to 95% of patients sensitized to birch respond. As a member of the pathogenesis-related PR 10 family, its natural function is implicated in plant defense, with a member of the PR10 family being reported to be upregulated under iron deficiency. As such, we assessed the function of Bet v 1 to sequester iron and its immunomodulatory properties on human immune cells. Binding of Bet v 1 to iron quercetin complexes FeQ2 was determined in docking calculations and by spectroscopy. Serum IgE-binding to Bet v 1 with (holoBet v1) and without ligands (apoBet v 1) were assessed by ELISA, blocking experiments and Western Blot. Crosslinking-capacity of apo/holoBet v 1 were assessed on human mast cells and Arylhydrocarbon receptor (AhR) activation with the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for labile iron and phenotypic changes by flow cytometry. Bet v 1 bound to FeQ2 strongly with calculated Kd values of 1 nm surpassing affinities to quercetin alone nearly by a factor of 1000. Binding to FeQ2 masked IgE epitopes and decreased IgE binding up to 80% and impaired degranulation of sensitized human mast cells. Bet v 1 facilitated the shuttling of quercetin, which activated the anti-inflammatory AhR pathway and increased the labile iron pool of human monocytic cells. The increase of labile iron was associated with an anti-inflammatory phenotype in CD14+monocytes and downregulation of HLADR. To summarize, we reveal for the first time that FeQ2 binding reduces the allergenicity of Bet v 1 due to ligand masking, but also actively contributes anti-inflammatory stimuli to human monocytes, thereby fostering tolerance. Nourishing immune cells with complex iron may thus represent a promising antigen-independent immunotherapeutic approach to improve efficacy in allergen immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false‐positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens. Objectives: In this study, we characterized a novel SWP allergen, Bomb m 4, a 30‐kDa lipoprotein, and evaluated its diagnostic sensitivity. Methods: Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27‐kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects. Results: rBomb m 4‐specific IgE was recognized by all 17 SWP allergic patients. The 27‐kDa glycoprotein and Bomb m 1 sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4 M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self‐inhibition of rBomb m 4 sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81–95, 191–205 and 224–238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27‐kDa glycoprotein and Bomb m 3. Conclusion: Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy. [ABSTRACT FROM AUTHOR]

Background: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5‐specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. Methods: A Phl p 5‐specific ELISA system was assessed with respect to accuracy, precision, inter‐assay (within laboratory) and inter‐laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. Results: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter‐assay variation (max. GCV 24%) and especially inter‐laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Conclusions: Based on the collaborative study results, the assessed Phl p 5‐specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method. [ABSTRACT FROM AUTHOR]

Progressive knowledge of allergenic structures resulted in a broad availability of allergenic molecules for diagnosis. Component‐resolved diagnosis allowed a better understanding of patient sensitization patterns, facilitating allergen immunotherapy decisions. In parallel to the discovery of allergenic molecules, there was a progressive development of a regulation framework that affected both in vitro diagnostics and Allergen Immunotherapy products. With a progressive understanding of underlying mechanisms associated to Allergen immunotherapy and an increasing experience of application of molecular diagnosis in daily life, we focus in analyzing the evidences of the value provided by molecular allergology in daily clinical practice, with a focus on Allergen Immunotherapy decisions. [ABSTRACT FROM AUTHOR]

Bet v 1 is the major allergen in birch pollen to which up to 95% of patients sensitized to birch respond. As a member of the pathogenesis-related PR 10 family, its natural function is implicated in plant defense, with a member of the PR10 family being reported to be upregulated under iron deficiency. As such, we assessed the function of Bet v 1 to sequester iron and its immunomodulatory properties on human immune cells. Binding of Bet v 1 to iron quercetin complexes FeQ2 was determined in docking calculations and by spectroscopy. Serum IgE-binding to Bet v 1 with (holoBet v1) and without ligands (apoBet v 1) were assessed by ELISA, blocking experiments and Western Blot. Crosslinking-capacity of apo/holoBet v 1 were assessed on human mast cells and Arylhydrocarbon receptor (AhR) activation with the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for labile iron and phenotypic changes by flow cytometry. Bet v 1 bound to FeQ2 strongly with calculated Kd values of 1 nm surpassing affinities to quercetin alone nearly by a factor of 1000. Binding to FeQ2 masked IgE epitopes and decreased IgE binding up to 80% and impaired degranulation of sensitized human mast cells. Bet v 1 facilitated the shuttling of quercetin, which activated the anti-inflammatory AhR pathway and increased the labile iron pool of human monocytic cells. The increase of labile iron was associated with an anti-inflammatory phenotype in CD14+monocytes and downregulation of HLADR. To summarize, we reveal for the first time that FeQ2 binding reduces the allergenicity of Bet v 1 due to ligand masking, but also actively contributes anti-inflammatory stimuli to human monocytes, thereby fostering tolerance. Nourishing immune cells with complex iron may thus represent a promising antigen-independent immunotherapeutic approach to improve efficacy in allergen immunotherapy.

The topic of standardization in relation to allergen products has been discussed by allergists, regulators, and manufacturers for a long time. In contrast to synthetic medicinal products, the natural origin of allergen products makes the necessary comparability difficult to achieve. This holds true for both aspects of standardization: Batch-to-batch consistency (or product-specific standardization) and comparability among products from different manufacturers (or cross-product comparability). In this review, we focus on how the United States and the European Union have tackled the topic of allergen product standardization in the past, covering the early joint standardization efforts in the 1970s and 1980s as well as the different paths taken by the two players thereafter until today. So far, these two paths have been based on rather classical immunological methods, including the corresponding benefits like simple feasability. New technologies such as mass spectrometry present an opportunity to redefine the field of allergen standardization in the future.

The topic of standardization in relation to allergen products has been discussed by allergists, regulators, and manufacturers for a long time. In contrast to synthetic medicinal products, the natural origin of allergen products makes the necessary comparability difficult to achieve. This holds true for both aspects of standardization: Batch-to-batch consistency (or product-specific standardization) and comparability among products from different manufacturers (or cross-product comparability). In this review, we focus on how the United States and the European Union have tackled the topic of allergen product standardization in the past, covering the early joint standardization efforts in the 1970s and 1980s as well as the different paths taken by the two players thereafter until today. So far, these two paths have been based on rather classical immunological methods, including the corresponding benefits like simple feasability. New technologies such as mass spectrometry present an opportunity to redefine the field of allergen standardization in the future. [ABSTRACT FROM AUTHOR]

One hundred thirty-one assays were performed to determine the level of specific IgE to major molecular components of the birch pollen—rBet v 1, timothy grass pollen—rPhl p1, rPhl p 5b, and house dust mites—nDer p1, rDer p2 as well as to the minor components of birch pollen—rBet v 2, rBet v 4, timothy grass pollen—rPhl p7, rPhl p 12, and house dust mites rDer p10 in patients with asthma (N = 66) and allergic rhinitis (N = 65). It is well established that sensitization to major and minor allergens facilitates the selection of an allergen for the allergen-specific immunotherapy (ASIT). According to the concept of molecular-based allergen diagnostics, the ASIT efficacy is expected to be high when IgЕ to major components are present and antibodies to minor components are absent. When elevated IgЕ levels to major and minor ASIT proteins are present, a moderate ASIT efficacy is expected. [ABSTRACT FROM AUTHOR]