In silico approaches for the identification of potential allergens among hypothetical proteins from Alternaria alternata and its functional annotation.

Direct exposure to the fungal species Alternaria alternata is a major risk factor for the development of asthma, allergic rhinitis, and inflammation. As of November 23rd 2020, the NCBI protein database showed 11,227 proteins from A. alternata genome as hypothetical proteins (HPs). Allergens are the main causative of several life-threatening diseases, especially in fungal infections. Therefore, the main aim of the study is to identify the potentially allergenic inducible proteins from the HPs in A. alternata and their associated functional assignment for the complete understanding of the complex biological systems at the molecular level. AlgPred and Structural Database of Allergenic Proteins (SDAP) were used for the prediction of potential allergens from the HPs of A. alternata. While analyzing the proteome data, 29 potential allergens were predicted by AlgPred and further screening in SDAP confirmed the allergic response of 10 proteins. Extensive bioinformatics tools including protein family classification, sequence-function relationship, protein motif discovery, pathway interactions, and intrinsic features from the amino acid sequence were used to successfully predict the probable functions of the 10 HPs. The functions of the HPs are characterized as chitin-binding, ribosomal protein P1, thaumatin, glycosyl hydrolase, and NOB1 proteins. The subcellular localization and signal peptide prediction of these 10 proteins has further provided additional information on localization and function. The allergens prediction and functional annotation of the 10 proteins may facilitate a better understanding of the allergenic mechanism of A. alternata in asthma and other diseases. The functional domain level insights and predicted structural features of the allergenic proteins help to understand the pathogenesis and host immune tolerance. The outcomes of the study would aid in the development of specific drugs to combat A. alternata infections.

Identification, Characterization, Cloning, and Cross-Reactivity of Zan b 2, a Novel Pepper Allergen of 11S Legumin.

Protein from Sichuan peppers can elicit mild to severe allergic reactions. However, little is known about their allergenic proteins. We aimed to isolate, identify, clone, and characterize Sichuan pepper allergens and to determine its allergenicity and cross-reactivities. Sichuan pepper seed proteins were extracted and then analyzed by SDS-PAGE. Western blotting was performed with sera from Sichuan pepper-allergic individuals. Proteins of interest were purified using hydrophobic interaction chromatography and gel filtration and further analyzed by analytical ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry (MS). Their coding region was amplified in the genome. IgE reactivity and cross-reactivity of allergens were evaluated by dot blot, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot showed IgE binding to a 55 kDa protein. This protein was homologous to the citrus proteins and has high stability and a sheet structure. Four DNA sequences were cloned. Six patients' sera (60%) showed specific IgE reactivity to this purified 11S protein, which was proved to have cross-reactivation with extracts of cashew nuts, pistachios, and citrus seeds. A novel allergen in Sichuan pepper seeds, Zan b 2, which belongs to the 11S globulin family, was isolated and identified. Its cross-reactivity with cashew nuts, pistachios, and citrus seeds was demonstrated.

Identification and characterization of a novel nematode pan allergen (NPA) from Wuchereria bancrofti and their potential role in human filarial tropical pulmonary eosinophilia (TPE).

BACKGROUND: Tropical pulmonary eosinophilia (TPE) is a chronic respiratory syndrome associated with Lymphatic Filariasis (LF), a tropical parasitic infection of the human, transmitted by mosquitoes. The larval form of LF (microfilariae) are trapped in the lungs of TPE subjects have a major role in initiating the TPE syndrome. To date, there are no reports on the potential allergen that is responsible for generating parasite-specific IgE in TPE. METHODOLOGY/PRINCIPAL FINDINGS: In this project, we screened a cDNA expression library of the microfilarial stages of Wuchereria bancrofti with monoclonal IgE antibodies prepared from subjects with clinical filarial infections. Our studies identified a novel molecule that showed significant sequence similarity to an allergen. A blast analysis showed the presence of similar proteins in a number of nematodes parasites. Thus, we named this molecule as Nematode Pan Allergen (NPA). Subsequent functional analysis showed that NPA is a potent allergen that can cause release of histamine from mast cells, induce secretion of proinflammatory cytokines from alveolar macrophages and promote accumulation of eosinophils in the tissue, all of which occur in TPE lungs. CONCLUSIONS/SIGNIFICANCE: Based on our results, we conclude that the NPA protein secreted by the microfilariae of W. bancrofti may play a significant role in the pathology of TPE syndrome in LF infected individuals. Further studies on this molecule can help design an approach to neutralize the NPA in an attempt to reduce the pathology associated with TPE in LF infected subjects.

Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

Production of a Ric c3 hypo-allergen with no IgE binding or anaphylactogenic activity.

Several studies have been carried out to expand the use of Ricinus communis L. castor bean (Ricinus communis L castor bean.). This oilseed finds appropriate conditions for its development in Brazil, with more than 700 applications. The main allergens of this plant are Ric c1 and Ric c3, that cross-react with various aeroallergens and food allergens such as peanuts, soybeans, corn, and wheat. This study aimed to determine the effect of mutations in Ric c3 amino acid residues known to affect IgE binding and allergy challenges. Based on the Ric c3 structure, B-cell epitopes, and amino acid involved in IgE binding, we produce recombinant mutant protein, mrRic c3, secreted from E. coli. Strategic glutamic acid residues in IgE-biding regions were changed by Leucine. The allergenicity of mrRic c3 was evaluated by determination of IgE, IgG1, and total IgG in immunized Balb/c mice and by degranulation assays of mast cells isolated from Wistar rats. The mrRic c3 presented a percentage of mast cell degranulation close to that seen in the negative control, and the immunization of mice with mrRic c3 presented lower levels of IgE and IgG1 than the group treated with the protein without mutations. The mutant mrRic c3 had an altered structure and reduced ability to stimulate pro-inflammatory responses and bind IgE but retained its ability to induce blocking antibodies. Thus, producing a hypoallergenic mutant allergen (mrRic c3) may be essential in developing new AIT strategies.

rBet v 1a-BanLecwt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors.

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.

First nanoplate digital PCR method to trace allergenic foods: Improved sensitivity for the detection of sesame.

Sesame (Sesamum indicum L.) is an important allergenic food whose presence can be the cause of severe allergic reactions in sensitised individuals. In this work, nanoplate digital PCR (ndPCR) was used to develop two methods to detect trace amounts of sesame in processed foods and compared with previously proposed real-time PCR assays. Two independent ndPCR approaches were successfully advanced, achieving sensitivities of 5 and 0.1 mg/kg of sesame in dough/biscuits, targeting the CO6b-1 and ITS regions, respectively. The sensitivity using both targets was improved by one order of magnitude comparing with real-time PCR and was not affected by food processing. CO6b-1 system was not influenced by food matrix, exhibiting similar performance regardless the use of complex matrix extracts or serial diluted DNA. Herein, ndPCR was proposed for the first time for the detection of allergenic foods with the advantage of providing better performance than real-time PCR regarding sensitivity and robustness.

Chromosome-level genome of Ambrosia trifida provides insights into adaptation and the evolution of pollen allergens.

Ambrosia trifida (giant ragweed) is an invasive plant that can cause serious damage to natural ecosystems and severe respiratory allergies. However, the genomic basis of invasive adaptation and pollen allergens in Ambrosia species remain largely unknown. Here, we present a 1.66 Gb chromosome-scale reference genome for giant ragweed and identified multiple types of genome duplications, which are responsible for its rapid environmental adaptation and pollen development. The largest copies number and species-specific expansions of resistance-related gene families compared to Heliantheae alliance might contribute to resist stresses, pathogens and rapid adaptation. To extend the knowledge of evolutionary process of allergic pollen proteins, we predicted 26 and 168 potential pollen allergen candidates for giant ragweed and other Asteraceae plant species by combining machine learning and identity screening. Interestingly, we observed a specific tandemly repeated array for potential allergenic pectate lyases among Ambrosia species. Rapid evolutionary rates on putative pectate lyase allergens may imply a crucial role of nonsynonymous mutations on amino acid residues for plant biological function and allergenicity. Altogether, this study provides insight into the molecular ecological adaptation and putative pollen allergens prediction that will be helpful in promoting invasion genomic research and evolution of putative pollen allergy in giant ragweed.

Genomics of Shrimp Allergens and Beyond.

Allergy to shellfishes, including mollusks and crustaceans, is a growing health concern worldwide. Crustacean shellfish is one of the "Big Eight" allergens designated by the U.S. Food and Drug Administration and is the major cause of food-induced anaphylaxis. Shrimp is one of the most consumed crustaceans triggering immunoglobulin E (IgE)-mediated allergic reactions. Over the past decades, the allergen repertoire of shrimp has been unveiled based on conventional immunodetection methods. With the availability of genomic data for penaeid shrimp and other technological advancements like transcriptomic approaches, new shrimp allergens have been identified and directed new insights into their expression levels, cross-reactivity, and functional impact. In this review paper, we summarize the current knowledge on shrimp allergens, as well as allergens from other crustaceans and mollusks. Specific emphasis is put on the genomic information of the shrimp allergens, their protein characteristics, and cross-reactivity among shrimp and other organisms.

Predicting the allergenic risk of Phosphite-NAD+-Oxidoreductase and purple acid phosphatase 17 proteins in genetically modified canola using bioinformatic approaches.

Recent advancements in the generation of high-throughput multi-omics data have provided a vast array of candidate genes for the genetic engineering of plants. However, as part of their safety assessment, newly expressed proteins in genetically modified crops must be evaluated for potential cross-reactivity with known allergens. In this study, we developed transgenic canola plants expressing the Arabidopsis thaliana PAP17 gene and a novel selectable marker composed of the ptxD gene from Pseudomonas stutzeri. To evaluate the potential allergenic cross-reactivity of the AtPAP17 and PTXD proteins expressed in transgenic canola, we applied a comprehensive approach utilizing sequence-based, motif-based, and 3D structure-based analyses. Our results demonstrate that the risk of conferring cross-reactivity with known allergens is negligible, indicating that the expression of these proteins in transgenic canola poses a low allergenic risk.

Identification of Pla a 7 as a novel pollen allergen group in Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is recognized as a source of allergenic pollen worldwide. Currently, five Platanus acerifolia pollen allergens belonging to different protein families have been identified, in which profilin and enolase were characterized by our group recently. Besides, we also screened and identified a novel allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens. However, the role of this novel allergen group in Platanus acerifolia pollen allergy was unclear. Therefore, we further investigated the allergenicity and clarify its clinical relevance in this study. METHODS: The natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test. RESULTS: A novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients' sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test. CONCLUSIONS: Triosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy.

Digestion-Resistant Linear Epitopes as Dominant Contributors to Strong Allergenicity of Tropomyosin in Antarctic Krill (Euphausia superba).

Although tropomyosin has been identified as a major allergen in Antarctic krill, the digestive fate of Antarctic krill tropomyosin and its relationship with allergenicity are unknown. In this study, Antarctic krill tropomyosin was administered to BALB/c mice via both gavage and intraperitoneal injection to explore its sensitizing and eliciting capacity, and its digestion products were analyzed for structural changes and digestion-resistant linear epitopes. Mice gavaged with tropomyosin exhibited lower levels of specific IgE and IgG1, mast cell degranulation, vascular permeability, and anaphylaxis symptoms than those in the intraperitoneal injection group. This may be due to the destruction of macromolecular aggregates, loose expansion of the tertiary structure, complete disappearance of α-helix, and significant changes in molecular force upon the digestion of tropomyosin. Nevertheless, the intragastric administration of Antarctic krill tropomyosin still triggered strong allergic reactions, which was attributed to the existence of seven digestion-resistant linear epitopes (Glu26-His44, Thr111-Arg125, Glu157-Glu164, Glu177-Gly186, Val209-Ile225, Arg244-Arg255, and Val261-Ile270).

Quantitative Real-Time PCR for the Detection of Allergenic Species in Foods.

Food allergy is an increasing challenge to public health, with widespread global distribution. With no cure for this pathology, the food-allergic individuals are forced to adopt food eviction measurements, relying on label information to avoid consuming the offending foods. To safeguard these individuals, the analytical methods based on real-time PCR approaches are currently faced as excellent tools to verify labeling compliance, aiding industry and regulatory agencies to efficiently manage food allergen control programs. Therefore, this chapter intends to describe a protocol of real-time PCR to analyze allergenic food species. For method development, the main steps to be considered are (i) in silico sequence analysis and primer/hydrolysis probe design, (ii) preparation of calibrators (model foods containing the allergenic ingredient), (iii) efficient DNA extraction from complex food matrices, (iv) amplification by real-time PCR with hydrolysis probe (90-200 bp) targeting a highly specific DNA region (allergen-encoding gene), (v) sequencing PCR products for identity confirmation, and (vi) validation and application to commercial foods. Herein, a real-time PCR approach for the detection and quantification of cashew nut as an allergenic food is described as an example protocol, including all the steps for method development and validation.

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control Anopheles gambiae the mosquito vector of human malaria.

BACKGROUND: Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity. METHODS: Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived. RESULTS: While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible. CONCLUSIONS: Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.

Impact of Semiochemicals Binding to Fel d 1 on Its 3D Conformation and Predicted B-Cell Epitopes Using Computational Approaches.

The major cat allergen Fel d 1 is a tetrameric glycoprotein from the secretoglobin superfamily. Fel d 1's biological role is unknown, but it has been previously shown that it participates in semiochemical binding/transportation. Fel d 1 has linear epitopes, but its conformational epitope sites remain unclear. In this study, we predicted the B-cell epitopes of Fel d 1 and explored semiochemical dynamics with epitopes using bioinformatics tools. The epitope residues were tabulated for chains 1 and 2 and the heterodimers of Fel d 1. The residual interactions of Fel d 1 with IgE were evaluated, and the prominent epitope sites were predicted. The molecular dynamics simulation (MDS) of Fel d 1 was performed with seven reported semiochemicals to evaluate the Fel d 1-ligand complex stability and decipher the semiochemical effect on Fel d 1 conformational epitopes. Fel d 1-lauric acid, Fel d 1-oleic acid, and Fel d 1-progesterone showed more stability and less fluctuation than other compounds. Fel d 1-linoleic acid and Fel d 1-pregnenolone displayed the most unstable complex with fluctuations. The effects of conformational changes on epitopes are discussed. All the ligand complexes drive substantial fluctuation towards the functionally exposed IgE-binding epitopes. Fel d 1 could be examined for its ligand-binding and conformational changes caused by mutations of B-cell epitopes.

Filling Analytical Gaps in Allergen Detection─Real-Time PCR for the Detection of Commercially Relevant Cephalopods and Gastropods in Food.

Mollusks belong to the group of shellfish, which are considered to be among the elicitors of severe food allergies worldwide. In recent years, numerous PCR detection methods have been developed for other shellfish such as crustaceans. However, cephalopods and gastropods were not considered in the development of these shellfish detection systems. In this study, we have developed highly specific real-time PCR methods for the comprehensive detection of all commercially relevant cephalopod species and the gastropod families Helicidae, Buccinidae, and Muricidae in food matrices. In total, we cross-tested over 100 animal and plant species to show the specificity of our systems. The limit of detection (LOD12) was set at 1 pg of cephalopod and gastropod DNA or 10 ppm (mg/kg) spiked in a vegetarian food product. The robustness of the protocol was confirmed by testing multiple parameters while cooking and autoclaving of samples ensured the practical applicability of the systems.

Identification of key genes and the pathophysiology associated with allergen-specific immunotherapy for allergic rhinitis.

BACKGROUND: Allergen-specific immunotherapy (AIT) is a causative treatment in allergic rhinitis (AR), comprising long-term allergen administration and over three years of treatment. This study is carried out for revealing the mechanisms and key genes of AIT in AR. METHODS: The present study utilized online Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE37157 and GSE29521 to analyze the hub genes changes related to AIT in AR. Based on limma package, differential expression analysis for the two groups (samples of allergic patients prior to AIT and samples of allergic patients undergoing AIT) was performed to obtain differentially expressed genes (DEGs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs were conducted using DAVID database. A Protein-Protein Interaction network (PPI) was built and a significant network module was acquired by using Cytoscape software (Cytoscape, 3.7.2). Utilizing the miRWalk database, we identified potential gene biomarkers, constructed interaction networks of target genes and microRNAs (miRNAs) using Cytoscape software, and explore the cell type-specific expression patterns of these genes in peripheral blood using publicly available single-cell RNA sequencing data (GSE200107). Finally, we are using PCR to detect changes in the hub genes that are screened using the above method in peripheral blood before and after AIT treatment. RESULTS: GSE37157 and GSE29521 included 28 and 13 samples, respectively. A total of 119 significantly co-upregulated DEGs and 33 co-downregulated DEGs were obtained from two datasets. The GO and KEGG analyses demonstrated that protein transport, positive regulation of apoptotic process, Natural killer cell mediated cytotoxicity, T cell receptor signaling pathway, TNF signaling pathway, B cell receptor signaling pathway and Apoptosis may be potential candidate therapeutic targets for AIT of AR. From the PPI network, 20 hub genes were obtained. Among them, the PPI sub-networks of CASP3, FOXO3, PIK3R1, PIK3R3, ATF4, and POLD3 screened out from our study have been identified as reliable predictors of AIT in AR, especially the PIK3R1. CONCLUSION: Our analysis has identified novel gene signatures, thereby contributing to a more comprehensive understanding of the molecular mechanisms underlying AIT in the treatment of AR.

Evolution of a Cockroach Allergen into the Major Protein of Termite Royal Jelly.

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.

Exosomes Derived from Dermatophagoides farinae Induce Allergic Airway Inflammation.

House dust mites (HDMs) are a major source of indoor allergens that cause airway allergic disease. Dermatophagoides farinae, a predominant species of HDMs in China, has demonstrated pathogenic role in allergic disorders. Exosomes derived from human bronchoalveolar lavage fluid have been strongly associated with allergic respiratory diseases progression. However, the pathogenic role of D. farinae-derived exosomes in allergic airway inflammation has remained unclear until now. Here, D. farinae was stirred overnight in phosphate-buffered saline, and the supernatant was used to extract exosomes by ultracentrifugation. Then, shotgun liquid chromatography-tandem mass spectrometry and small RNA sequencing were performed to identify proteins and microRNAs contained in D. farinae exosomes. Immunoblotting, Western blotting, and enzyme-linked immunosorbent assay demonstrated the specific immunoreactivity of D. farinae-specific serum IgE antibody against D. farinae exosomes, and D. farinae exosomes were found to induce allergic airway inflammation in a mouse model. In addition, D. farinae exosomes invaded 16-HBE bronchial epithelial cells and NR8383 alveolar macrophages to release the inflammation-related cytokines interleukin-33 (IL-33), thymic stromal lymphopoietin, tumor necrosis factor alpha, and IL-6, and comparative transcriptomic analysis of 16-HBE and NR8383 cells revealed that immune pathways and immune cytokines/chemokines were involved in the sensitization of D. farinae exosomes. Taken together, our data demonstrate that D. farinae exosomes are immunogenic and may induce allergic airway inflammation via bronchial epithelial cells and alveolar macrophages. IMPORTANCE Dermatophagoides farinae, a predominant species of house dust mites in China, has displayed pathogenic role in allergic disorders, and exosomes derived from human bronchoalveolar lavage fluid have been strongly associated with allergic respiratory diseases progression. However, the pathogenic role of D. farinae-derived exosomes in allergic airway inflammation has remained unclear until now. This study, for the first time, extracted exosomes from D. farinae, and sequenced their protein cargo and microRNAs using shotgun liquid chromatography-tandem mass spectrometry and small RNA sequencing. D. farinae-derived exosomes trigger allergen-specific immune responses and present satisfactory immunogenicity, as revealed by immunoblotting, Western blotting, and enzyme-linked immunosorbent assay and may induce allergic airway inflammation via bronchial epithelial cells and alveolar macrophages. Our data provide insights into the mechanisms of allergic airway inflammation caused with D. farinae-derived exosomes and the treatment of house dust mite-induced allergic airway inflammation.

Immunological comparison of recombinant shrimp allergen Pen m 4, produced in Pichia pastoris and Escherichia coli.

Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3-2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.