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BACKGROUND: A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. OBJECTIVE: We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. METHODS: A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. RESULTS: The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. CONCLUSION: An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.
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Alérgenos/genética , Antígenos de Dermatophagoides/genética , Dermatophagoides farinae/genética , Dermatophagoides farinae/imunologia , Genoma , Transcriptoma , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Dermatophagoides farinae/anatomia & histologia , Dermatophagoides farinae/classificação , Dermatophagoides farinae/microbiologia , Feminino , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Metagenoma , Microbiota , Filogenia , ProteômicaRESUMO
Background: House dust mites (HDMs), including Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) species, represent a major source of inhalant allergens that induce IgE-mediated anaphylactic reactions. HDM allergen identification is important to the diagnosis and treatment of allergic diseases. Here, we report the identification of a novel HDM allergen, which we suggest naming Der f 40, and its immunodominant IgE epitopes. Methods: The recombinant protein Der f 40 was expressed using a pET prokaryotic expression system and purified with Ni-NTA resins. IgE binding activity was evaluated by IgE-western blot, dot-blot, and ELISA. Mast cell activation testing was performed to assess the cellular effects of IgE binding in mouse bone marrow derived mast cells (BMMCs) expressing human FcεRI. IgE binding assays were performed with truncated and hybrid Der f 40 protein molecules to find immunodominant IgE epitopes. Results: A 106-amino acid (aa) recombinant Der f Group 40 protein (rDer f 40) was obtained (GenBank accession No. XP_046915420.1) as thiredoxin-like protein. Der f 40 was shown to bind IgE from HDM allergic serum in vitro (9.68%; 12/124 in IgE-ELISA), and shown to promote the release of ß-hexosaminidase from BMMCs dose-dependently when administered with HDM allergic sera. The Der f Group 40 protein was named Der f 40 and listed in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. IgE binding assays with Der f 40-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The 76-106-aa region of C-terminus was identified as an immunodominant IgE epitope of Der f 40. Conclusion: A novel HDM allergen with robust IgE binding activity was identified and named Der f 40. An immunodominant IgE epitope of Der f 40 with conformational dependency was identified in the C-terminus (aa 76-106). These findings provide new information that may be useful in the development of diagnostic and therapeutic agents for HDM allergy.
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Immunoglobulin (Ig)E-associated mast cell (MC) activation triggers pro-inflammatory signals that underlie type I allergic diseases. Here, we examined the effects of the natural isoflavone formononetin (FNT) on IgE-mediated MC activation and associated mechanisms of high-affinity IgE receptor (FcεRI) signal inhibition. The effects of FNT on the mRNA expression of inflammatory factors, release of histamine and ß-hexosaminidase (ß-hex), and expression of signaling proteins and ubiquitin (Ub)-specific proteases (USPs) were analyzed in two sensitized/stimulated MC lines. FcεRIγ-USP interactions were detected by co-immunoprecipitation (IP). FNT dose-dependently inhibited ß-hex activity, histamine release, and inflammatory cytokine expression in FcεRI-activated MCs. FNT suppressed IgE-induced NF-κB and MAPK activity in MCs. The oral administration of FNT attenuated passive cutaneous anaphylaxis (PCA) reactions and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) reactions in mice. FNT reduced the FcεRIγ chain expression, via increased proteasome-mediated degradation, and induced FcεRIγ ubiquitination by inhibiting USP5 and/or USP13. FNT and USP inhibition may be useful for suppressing IgE-mediated allergic diseases.
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Anafilaxia , Isoflavonas , Camundongos , Animais , Receptores de IgE/genética , Receptores de IgE/metabolismo , Mastócitos , Transdução de Sinais , Anafilaxia/tratamento farmacológico , Imunoglobulina E/metabolismo , Isoflavonas/farmacologia , Isoflavonas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Degranulação CelularRESUMO
Background: House dust mites (HDMs) are the main source of indoor inhalatory allergens that cause IgE-mediated allergic diseases. The discovery and identification of HDM allergens are important for the diagnosis and treatment of allergic diseases. Objective: We sought to identify a Group 39 Dermatophagoides pteronyssinus (Der p) allergen, namely Der p 39, and explore its immunodominant IgE epitopes. Methods: Homology analysis of amino acid (aa) sequences in HDM and human troponin C (TnC)-like protein was performed. Total RNA of Der p was extracted and used to amplify Der p 39 cDNA with specific primers. Recombinant Der p 39 protein was expressed with a pET-His prokaryotic expression system and purified with Ni-NTA resins. IgE binding was evaluated with western blot, dot blot, and enzyme-linked immunosorbent assay (ELISA) experiments. The IgE binding epitopes of Der p 39 were identified by observing HDM-allergic sera interactions with truncated and hybrid proteins formed from Der p 39 and human TnC-like proteins. Results: The Der p 39 open reading frame (ORF) cDNA was found to be 462 base pairs and registered in the NCBI library (GenBank no. MZ336019.1). Der p 39, which encoded 153 aa, was found to have 35.63% and 99.35% homology with human TnC and Dermatophagoides farina (Der f) 39, respectively. IgE-ELISA showed IgE binding with expressed and purified recombinant Der p 39 (18 kDa) in 5/87 (5.75%) HDM-allergic sera samples. Analyses of IgE binding with Der p 39-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The data from this study were submitted to the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature database. Conclusion: Der p 39 was identified as a minor HDM allergen with a conformational IgE binding epitope. These findings could have important theoretical implications in the development of HDM allergy diagnostics and therapeutics.
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Mast cells (MCs) are the main effector cells in the onset of high-affinity receptor for IgE (FcεRI)-mediated allergic diseases. The aim of this study was to test whether dihydrocoumarin (DHC), a food flavoring agent derived from Melilotus officinalis, can block IgE-induced MC activation effects and to examine the potential molecular mechanisms by which DHC affects MC activation. Rat basophilic leukemia cells (RBLs) and mouse bone marrow-derived mast cells (BMMCs) were sensitized with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated with DNP-human serum albumin antigen, and treated with DHC. Western blot analyses were performed to detect the expression of signaling proteins. Murine IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) models were used to examine DHC effects on allergic reactions in vivo. DHC inhibited MC degranulation, as evidenced by reduced ß-hexosaminidase activity and histamine levels, and reduced morphological changes associated with MC activation, namely cellular elongation and F-actin reorganization. DHC inhibited the activation of MAPK, NF-κB, and AP-1 pathways in IgE-activated MCs. Additionally, DHC could attenuate IgE/Ag-induced allergic reactions (dye extravasation and ear thickening) in PCA as well as OVA challenge-induced reactions in ASA mice (body temperature, serum histamine and IL-4 secretion changes). In conclusion, DHC suppressed MC activation. DHC may represent a new MC-suppressing treatment strategy for the treatment of IgE-mediated allergic diseases.
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Anafilaxia , Mastócitos , Anafilaxia/tratamento farmacológico , Animais , Degranulação Celular , Aromatizantes/metabolismo , Imunoglobulina E/metabolismo , Inflamação/metabolismo , Camundongos , Anafilaxia Cutânea Passiva , RatosRESUMO
Accurate house dust mite (HDM) genome and transcriptome data would promote our understanding of HDM allergens. We sought to assemble chromosome-level genome and precise transcriptome profiling of Dermatophagoides farinae and identify novel allergens. In this study, genetic material extracted from HDM bodies and eggs were sequenced. Short-reads from next generation sequencing (NGS) and long-reads from PacBio/Nanopore sequencing were used to construct the D. farinae nuclear genome, transcriptome, and mitochondrial genome. The candidate homologs were screened through aligning our assembled transcriptome data with amino acid sequences in the WHO/IUIS database. Our results showed that compared with the D. farinae draft genome, bacterial DNA content in the presently developed sequencing reads was dramatically reduced (from 22.9888% to 1.5585%), genome size was corrected (from 53.55 Mb to 58.77 Mb), and the contig N50 was increased (from 8.54 kb to 9365.49 kb). The assembled genome has 10 contigs with minimal microbial contamination, 33 canonical allergens and 2 novel allergens. Eight homologs (≥50% homology) were cloned; 2 bound HDM allergic-sera and were identified as allergens (Der f 37 and Der f 39). In conclusion, a chromosome-level genome, transcriptome and mitochondrial genome of D. farinae was generated to support allergen identification and development of diagnostics and immunotherapeutic vaccines.
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Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response to Aspergillus fumigatus (Af) exposure, which induces a strong T helper 2 (Th2) response via mechanisms that have yet to be elucidated. The aim of the present study was to investigate the hypothesis that T2 ribonuclease from Af (Af RNASET2) induces M2type macrophage polarization to produce a T helper 2 (Th2) immune response. Recombinant Af RNASET2 (rAf RNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAf RNASET2 for in vivo analyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAf RNASET2 in vitro using flow cytometry, reverse transcriptionquantitative PCR, and western blot analysis. The results predicted that the mature Af RNASET2 protein (382 amino acids; GenBank no. MN593022) contained two conserved amino acid sequence (CAS) domains, termed CAS1 and CAS2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2related cytokines interleukin (IL)4, IL10, and IL13 were upregulated in mice immunized with rAf RNASET2. RAW264.7 macrophages treated with rAf RNASET2 showed increased mRNA expression levels of M2 factors [arginase 1, Il10, and Il13]; however, there was no difference in cells treated with rAf RNASET2 that had been inactivated with a ribonuclease inhibitor (RNasin). The protein expression levels of IL10 in macrophage culture supernatant were also increased following stimulation with rAf RNASET2. In addition, rAf RNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAf RNASET2induced IL10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.
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Aspergillus fumigatus/enzimologia , Endorribonucleases/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Células Th2/citologia , Células Th2/metabolismo , Animais , Endorribonucleases/genética , Feminino , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Células Th2/imunologiaRESUMO
The detection of allergenspecific immunoglobulin (Ig)E is an important method for the diagnosis of IgEmediated allergic diseases. The sensitivity of the indirect IgEELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts. To overcome these limitations, a novel capture IgEELISA based on a recombinant Der f 1/Der f 2 fusion protein (rDer f 1/2) was developed to enhance the sensitivity to IgEs that bind allergens from the house dust mite (HDM) species Dermatophagoides farina. pET28Der f 1/2 was constructed and expressed in Escherichia coli BL21 (DE3) pLysS. The purified fusion protein was evaluated by IgE western blotting, IgE dot blotting and indirect IgEELISA. CaptureELISA was performed by coating wells with omalizumab and incubating in series with sera, biotinylated Der f 1/2, horseradish peroxidaseconjugated streptavidin and 3,3,5,5tetramethylbenzidine. The relative sensitivities of indirectELISA and captureELISA for HDM allergenspecific IgE binding were determined; sera from nonallergic individuals were used as the control group. rDer f 1/2 was expressed in the form of inclusion bodies comprising refolded protein, which were then purified. It exhibited increased IgEspecific binding (24/28, 85.8%) than rDer f 1 (21/28, 75.0%) or rDer f 2 (22/28, 78.6%) with HDMallergic sera. Furthermore, in a random sample of HDMallergic sera (n=71), captureELISA (71/71, 100%) was more sensitive than indirectELISA (68/71, 95.8%) for the detection of HDMspecific IgEs (P<0.01), indicating that this novel method may be useful for the diagnosis of HDM allergy.
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Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina E/imunologia , Proteínas Recombinantes de Fusão , Adolescente , Adulto , Animais , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Ligação Proteica/imunologia , Pyroglyphidae/imunologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: The identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s). METHODS: Der p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed. RESULTS: We cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell ß-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes. CONCLUSIONS: We identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment.
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Previously, a ubiquinolcytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using NiNTA resins, and IgEbinding activity was determined using IgEwestern blotting and enzymelinked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgEdot blotting and IgEELISA with synthetic polypeptides and HDMallergic sera. Threedimensional (3D) structural modeling was used to analyze the position of the immunodominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgEELISA and western blot analysis showed that all of the Der fhuman UQCRB hybrid proteins generated, except for the one lacking 59 residues of the Nterminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the Nterminal reacted with IgEs from HDMallergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32aa Nterminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgEbinding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32residue region of the Nterminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.
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Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Dermatophagoides farinae/imunologia , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Pyroglyphidae/imunologia , Adolescente , Adulto , Animais , Proteínas de Artrópodes/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Hipersensibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ratos , Adulto JovemRESUMO
Allergic diseases are inflammatory disorders that involve many types of cells and factors, including allergens, immunoglobulin (Ig)E, mast cells, basophils, cytokines and soluble mediators. Among them, IgE plays a vital role in the development of acute allergic reactions and chronic inflammatory allergic diseases, making its control particularly important in the treatment of IgE-mediated allergic diseases. This review provides an overview of the current state of IgE targeted therapy development, focusing on three areas of translational research: IgE neutralization in blood; IgE-effector cell elimination; and IgE+ B cell reduction. IgE-targeted medicines such as FDA approved drug Xolair (Omalizumab) represent a promising avenue for treating IgE-mediated allergic diseases given the pernicious role of IgE in disease progression. Additionally, targeted therapy for IgE-mediated allergic diseases may be advanced through cellular treatments, including the modification of effector cells.
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Over the recent decades, China experienced several emerging virus outbreaks including those caused by the severe acute respiratory syndrome- (SARS-) coronavirus (Cov), H5N1 virus, and H7N9 virus. The SARS tragedy revealed faults in China's infectious disease prevention system, propelling the Chinese government to enact reforms that enabled better combating of the subsequent H1N1 and H7N9 avian flu epidemics. The system is buttressed by three fundamental, mutually reinforcing components: (1) enduring government administration reforms, including legislation establishing a unified public health emergency management system; (2) prioritized funding for biotechnology and biomedicine industrialization, especially in the areas of pathogen identification, drug production, and the development of vaccines and diagnostics; and (3) increasing investment for public health and establishment of a rapid-response infectious diseases prevention and control system. China is now using its hard-gained experience to support the fight against Ebola in Africa and the Middle East Respiratory Syndrome in its own country.