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1.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
2.
Int J Mol Sci ; 20(12)2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234267

RESUMO

The house dust mite (HDM) Dermatophagoides pteronyssinus is an important risk factor for asthma and rhinitis. Allergen specific immunotherapy that is based on recombinant proteins has been proposed for the safer and more efficient treatment of allergic diseases. The aim of this study was to design and obtain a hybrid protein (DPx4) containing antigenic regions of allergens Der p 1, Der p 2, Der p 7, and Der p 10 from this mite. DPx4 was produced in Escherichia coli and its folding was determined by circular dichroism. Non-denaturing dot-blot, ELISA, basophil activation test, dot blot with monoclonal antibodies, ELISA inhibition, and cysteine protease activity assays were performed. Mice that were immunized with DPx4 were also analyzed. We found that DPx4 had no cysteine protease activity and it showed significantly lower IgE reactivity than Der p 1, Der p 2, and D. pteronyssinus extract. DPx4 induced lower basophil activation than Der p 2 and the allergen extract. Immunized mice produced IgG antibodies that inhibited the binding of allergic patient's IgE to the allergen extract and induced comparatively higher levels of IL-10 than the extract in peripheral blood mononuclear cells (PBMC) culture. These results suggest that DPx4 has immunological properties that are useful for the development of a mite allergy vaccine.


Assuntos
Alérgenos/uso terapêutico , Antígenos de Dermatophagoides/uso terapêutico , Dermatophagoides pteronyssinus/imunologia , Hipersensibilidade/prevenção & controle , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Dermatophagoides pteronyssinus/genética , Feminino , Humanos , Hipersensibilidade/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico
3.
Mol Med Rep ; 19(5): 3497-3504, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896856

RESUMO

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.


Assuntos
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 Jovem
4.
Int Immunopharmacol ; 70: 216-224, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851701

RESUMO

OBJECTIVES: House dust mites, including Der p1, are common allergens. The current study was designed to explore the allergen-specific immune tolerance effects of Der p1-modified dendritic cells (DCs) through IL-4, IL-10 and IL-13 on an allergic rhinitis (AR) mouse model. METHODS: A lentivirus was modified to express Derp1. Then, immature DCs from mice were infected with this modified lentivirus to generate a lenti-Derp1-GFP DCs. 24 mice were random divided into four groups (n = 6 each), AR mouse were sensitized by Derp1 allergens and treated with lenti-GFP DCs (GFP-DC/AR group), or lenti-Derp1-GFP DCs (Der p1-DC/AR group) and dexamethasone (Dex/AR group), mice in the control group were treated with PBS instead of Der p1 then also intraperitoneally injected with 5 × 106 lenti-GFP DCs/mouse. AR symptoms expressed by each mouse were recorded. The proportions of CD4+CD25+Foxp3+ regulatory T cells among CD4+ T cells in the peripheral blood, and mRNA and protein expression levels of IL-4, IL-10, and IL-13 were measured. RESULTS: DCs infected with lenti-Derp1-GFP stimulated the maturation of DCs. Compared with the GFP-DC/AR group, mice in the Der p1-DC/AR group showed an ameliorated allergic response, a significant decrease in the levels of serum IgE, IgG1, and histamine, and a decrease in the expression of IL-4 and IL-13 mRNA and protein in the nasal mucosa. The expression of IL-10 increased in the Der p1-DC/AR group to a level similar to that observed in the Dex/AR group. CONCLUSIONS: These results indicate that Der p1-modified DCs have therapeutic potential for AR via downregulation of IL-4 and IL-13, and upregulation of IL-10.


Assuntos
Alérgenos/metabolismo , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Cisteína Endopeptidases/metabolismo , Células Dendríticas/fisiologia , Mucosa Nasal/fisiologia , Rinite Alérgica/imunologia , Alérgenos/genética , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Células Cultivadas , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Tolerância Imunológica/genética , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Lentivirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae/imunologia
5.
Int Arch Allergy Immunol ; 178(1): 10-18, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30380546

RESUMO

BACKGROUND: The aim of this work was to understand the molecular features that trigger the cross-reactivity observed between Der p 5 from Dermatophagoides pteronyssinus, Blo t 5 from Blomia tropicalis, and Der f 5 from D. farinae. METHODS: We collected serum from 60 house dust mite (HDM)-allergic patients residing in the Dellys area of Boumerdès province in northern Algeria. The presence of specific IgE to Der p 5, Der f 5, and Blo t 5 was analyzed. We performed in silico analysis of the structure of the different allergens in order to identify epitopes that can elicit the cross-reactivity of the sera. Synthetic peptides corresponding to the linear epitope sequence of Der p 5, Der f 5, and Blo t 5 were used to evaluate its implication in the cross-reactivity between the allergens. We also modified the sequence of the conformational epitope of Der p 5 by site-directed mutagenesis to mimic Blo t 5. RESULTS: Several sera of patients allergic to HDM contained specific IgE antibodies to Der p 5 and Blo t 5. We demonstrated that the linear epitope of Der p 5 and Blo t 5 is not involved in the cross-reactivity of the sera. Furthermore, mutations introduced in the sequence of Der p 5 to mimic Blo t 5 could not modulate the cross-reactivity between them. CONCLUSIONS: The major linear IgE epitopes of Der p 5 and Blo t 5 are involved in species-specific recognition. Our results may be useful for the development of a hypoallergenic vaccine against HDM group 5 allergens.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Reações Cruzadas/imunologia , Dermatophagoides pteronyssinus/imunologia , Epitopos/imunologia , Imunoglobulina E/imunologia , Adulto , Alérgenos/genética , Animais , Especificidade de Anticorpos , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Dermatophagoides pteronyssinus/genética , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Mutagênese , Proteínas Recombinantes , Adulto Jovem
6.
Protein Pept Lett ; 26(3): 184-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30526452

RESUMO

BACKGROUND: The dust mite Dermatophagoides farinae is a common worldwide cause of indoor allergies induced by its proteins, including the mid-tier allergen Der f 7. OBJECTIVE: To identify conformational epitopes in Der f 7 using mimotope mapping and computational modelling. METHODS: Here, we used standard hybridoma technology to generate 3 new monoclonal antibodies against Der f 7 and performed mimotope mapping by probing a random peptide phage display library. Computational tools, including Minox and the DiscoTope-2.0 Server were used to assess the structure and potential position of antigenic residues within Der f 7. RESULTS: Thirteen mimotopes sharing the common sequence --XX[LST]P[-E][LI]MLPLR[-S]- were identified. Further, computationally-predicted conformational epitopes were found at residues 1-7, 10, 27, 76-81, 92, and 130-133 of Der f 7, and the key amino acids for these epitopes were deduced to be 2P, 3I, 10E, 27E, 78E, 79E, 81I, 130S, and 132E based on the common mimotope sequence. CONCLUSION: We identified Der f 7 peptide mimotopes that may model binding sites for blocking antibodies. These may guide the development of immunotherapy for individuals with hypersensitivity to Der f 7.


Assuntos
Anticorpos Monoclonais , Antígenos de Dermatophagoides , Biblioteca de Peptídeos , Pyroglyphidae , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Pyroglyphidae/química , Pyroglyphidae/genética , Pyroglyphidae/imunologia
7.
Life Sci ; 213: 158-165, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30352241

RESUMO

Aim To investigate the immunomodulatory potential of a chimera composed of the receptor-binding domain of hemagglutinin 1 (H1s) from Influenza virus and Der p 2 (D2) allergen for allergen-specific immunotherapy of house-dust mite allergy (HDM). MAIN METHODS: H1sD2 chimera and D2 allergen were produced by genetic engineering in E. coli. Recombinant antigens were extracted from inclusion bodies by urea, then refolded and purified by immobilized- metal affinity chromatography (IMAC). Purity was verified by 2D-PAGE and secondary structures were assessed by CD spectroscopy. IgE reactivity of H1sD2 and D2 was tested in western blot with sera from 8 persons with clinical history of HDM allergy. Immunogenicity of H1sD2 and D2 were analyzed in Balb/c mice. Cytokine profile was analyzed by ELISA after stimulation of mouse spleen cells with H1sD2 and D2. Leukocyte population abundance of cells isolated from spleen and lymph node was assessed by flow cytometry. KEY FINDINGS: Purified recombinant proteins H1sD2 (42 kDa) and D2 (15 kDa) revealed well defined secondary structures, and preserved IgE reactive epitopes. Analysis of supernatants of mouse spleen cells after stimulation with H1sD2 and D2, revealed a qualitatively different cytokine profile from H1sD2 immunized mouse cells (increase in IL10). CD8+ cells were decreased in the lymph node of D2 immunized mice, whereas H1sD2 immunization led to an increase of CD8+ cells in both the lymph node and the spleen. SIGNIFICANCE: H1sD2 chimera attenuates Der p 2-inherent Th2 response and directs the immune response toward Th1 and Treg phenotype.


Assuntos
Alérgenos/uso terapêutico , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Hemaglutininas/metabolismo , Alérgenos/metabolismo , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Quimera , Epitopos/metabolismo , Escherichia coli/imunologia , Engenharia Genética/métodos , Hemaglutininas/genética , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Orthomyxoviridae/metabolismo , Pyroglyphidae/imunologia , Proteínas Recombinantes/genética
8.
Artigo em Chinês | MEDLINE | ID: mdl-30293259

RESUMO

Objective: To explore the therapeutic effects of dendritic cells (DC) modified by the dust-mite-allergen(Der p1) gene on mouse model of allergic rhinitis (AR). Methods: DC modified by the Der p1 gene (Der p1-DC) were prepared.Using random number table, 24 Balb\c mice were divided into four groups: immature DC (imDC)/AR group, dexamethasone/AR group, Der p1-DC/AR group and control group, with 6 mice in each group.AR mouse model was built with Der p1 and the mouse model of AR was established.The AR mice were respectively given by abdominal injection of Der p1-DC, imDC and dexamethasone.Normal control mice were treated with physiologic saline.ELISA method was used for determining the content of IgE, IgG1and histamine in blood.The relative expression of mRNA of IL-4 and IL-13 on nasal mucosa with protein was analyzed by RT-PCR and Westen blot methods.All the data were statistically analyzed by SPSS 19.0 statistical software, and the variance analysis was used in multiple groups of average samples. Results: The contents of IgE, IgG1 and histamine in the mice of Der p1-DC/AR group were lower than those in imDC/AR group ((0.560±0.110) OD 450 nm vs (1.150±0.280) OD 450 nm, (0.690±0.054) OD 450 nm vs (0.920±0.125) OD 450 nm, (4 145±670) pg/ml vs (7 685±669) pg/ml, t value was 4.80, 4.14, 9.16, respectively, all P<0.05), and the expression of IL-4 and IL-13 on nasal mucosa in Der p1-DC/AR group was remarkedly lower than those in imDC/AR group (0.41±0.25 vs 1.59±1.02, 0.26±0.01 vs 1.10±0.09, t value was 2.75, 22.72, respectively, all P<0.05). There was no statistically significant difference between the mice treated with Der p1-DC and dexamethasone group. Conclusions: The results showed that Der p1-DC could reduce inflammation in AR mice and decrease the expression of IL-4 and IL-13. It suggested that Der p1-DC can be used in the immunotherapy of AR mouse.


Assuntos
Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Cisteína Endopeptidases/genética , Células Dendríticas/imunologia , Poeira , Imunoterapia/métodos , Ácaros/imunologia , Rinite Alérgica/terapia , Animais , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Glucocorticoides/uso terapêutico , Histamina/sangue , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interleucina-13/genética , Interleucina-4/genética , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , RNA Mensageiro/sangue , Rinite Alérgica/sangue
9.
Mol Cell ; 70(2): 228-241.e5, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677491

RESUMO

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.


Assuntos
Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Sinalização do Cálcio , Movimento Celular , Mastócitos/metabolismo , Mucosa Nasal/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Pyroglyphidae/enzimologia , Receptores de Trombina/metabolismo , Serina Endopeptidases/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Antígenos de Dermatophagoides/efeitos adversos , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/efeitos adversos , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Asma/imunologia , Asma/metabolismo , Células HEK293 , Humanos , Exposição por Inalação , Inositol 1,4,5-Trifosfato/metabolismo , Ativação do Canal Iônico , Células Jurkat , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Mucosa Nasal/imunologia , Pyroglyphidae/genética , Pyroglyphidae/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Serina Endopeptidases/efeitos adversos , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia
10.
Braz J Med Biol Res ; 51(5): e6213, 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29561952

RESUMO

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.


Assuntos
Alérgenos/química , Antígenos de Dermatophagoides/química , Dermatophagoides farinae/química , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Tubulina (Proteína)/química , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Biologia Computacional , Dermatophagoides farinae/genética , Dermatophagoides farinae/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/genética , Estrutura Molecular , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia
11.
Immunol Lett ; 196: 103-112, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29408409

RESUMO

BACKGROUND: Sensitization to allergens of the house dust mites Dermatophagoides pteronyssinnus and Blomia tropicalis is an important risk factor for asthma and allergic diseases. Allergen-specific immunotherapy is currently based on natural allergen extracts, however, in the last years recombinant allergens with different modifications have shown promising immunological properties that may be advantageously applied for developing novel allergy vaccines. METHODS: A hybrid molecule (MAVAC-BD-2) containing epitopes of B. tropicalis (Blo t 5, Blo t 8 and Blo t 10) and D. pteronyssinus (Der p 1, Der p 2, Der p 7 and Der p 8) allergens was constructed, expressed in Escherichia coli and purified by affinity chromatography. Its folding was analyzed by circular dichroism. Antibody reactivities were evaluated by ELISA and non-denaturing dot blot assays using a battery of sera from mite allergic patients and non-allergic subjects. ELISA inhibition and dot blot assays with monoclonal antibodies were used to detect B-cell epitopes. Human basophil activation and induction of IgG-blocking antibodies in mice immunized with the hybrid protein were also evaluated. RESULTS: MAVAC-BD-2, expressed as a 22.8 kDa protein, showed a lower frequency and strength of IgE reactivity compared to Blo t 5, Der p 1, Der p 2 and the extracts of B. tropicalis and D. pteronyssinus. MAVAC-BD-2 inhibited 26% of IgE reactivity to Der p 2 and Blo t 5, reacted with anti-Der p 1 and anti-Der p 2 monoclonal antibodies and did not induce relevant basophil activation. MAVAC-BD-2 immunized mice produced specific antibodies that reacted against mite extracts and the purified allergens, as well as IgG antibodies that blocked the human IgE reactivity to mite extracts. CONCLUSION: MAVAC-BD-2 has hypoallergenic characteristics and in mice induces IgG antibodies that block the human IgE reactivity to mite extracts.


Assuntos
Alérgenos/imunologia , Proteínas de Artrópodes/imunologia , Dermatophagoides pteronyssinus/imunologia , Ácaros/imunologia , Proteínas Recombinantes de Fusão/imunologia , Alérgenos/genética , Alérgenos/metabolismo , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Reações Cruzadas/imunologia , Dermatophagoides pteronyssinus/genética , Dermatophagoides pteronyssinus/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunização , Imunoglobulina E/imunologia , Masculino , Camundongos Endogâmicos BALB C , Ácaros/genética , Ácaros/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
Int J Immunopathol Pharmacol ; 32: 394632017750997, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29357711

RESUMO

Airway epithelium cells are the first line of defense against airborne allergens. When cultured, epithelial cells can be exposed to various allergens, providing an ideal model to investigate allergic disorders. This study sought to characterize the profile of long noncoding (lnc) RNAs, which can regulate gene expression and exert functions in diverse cellular processes, in airway epithelial cells exposed to house dust mite allergens. NCI-H292 cells were exposed to house dust mite extract for 24 h. RNA expression was profiled in exposed and unexposed cells. There were 270 lncRNAs that were differentially expressed (fold change ≥ 2, P < 0.05) in NCI-H292 cells after stimulation with Dermatophagoides farinae (house dust mite) extracts. Furthermore, 119 lncRNAs and 22 messenger RNAs were co-expressed. Gene Ontology analysis showed that these under-regulated and up-regulated lncRNAs were associated with biological process, cellular component, and molecular function. After bioinformatic analysis of significantly regulated signaling pathways, we found these lncRNAs may target 16 gene pathways, including glycolysis, axon guidance, ErbB signaling, and mitogen-activated protein kinases (MAPK) signaling. The identification of differentially regulated lncRNAs in NCI-H292 cells after stimulation with Dermatophagoides farinae extracts, as well as their target gene pathways, can provide insight to the etiology and pathogenesis of allergy.


Assuntos
Alérgenos/biossíntese , Dermatophagoides farinae/metabolismo , RNA Longo não Codificante/biossíntese , Mucosa Respiratória/imunologia , Transcriptoma/fisiologia , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/biossíntese , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Linhagem Celular , Dermatophagoides farinae/genética , Dermatophagoides farinae/imunologia , Redes Reguladoras de Genes/fisiologia , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/imunologia , Mucosa Respiratória/metabolismo
15.
Braz. j. med. biol. res ; 51(5): e6213, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-889085

RESUMO

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.


Assuntos
Animais , Tubulina (Proteína)/química , Alérgenos/química , Epitopos de Linfócito T/química , Epitopos de Linfócito B/química , Dermatophagoides farinae/química , Antígenos de Dermatophagoides/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia , Alérgenos/genética , Alérgenos/imunologia , Estrutura Molecular , Estrutura Terciária de Proteína , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Epitopos de Linfócito B/genética , Biologia Computacional , Análise de Sequência de Proteína , Dermatophagoides farinae/genética , Dermatophagoides farinae/imunologia , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia
16.
Int J Mol Sci ; 18(6)2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28587273

RESUMO

Since the discovery that Der p 1 is a cysteine protease, the role of proteolytic activity in allergic sensitization has been explored. There are many allergens with proteolytic activity; however, exposure from dust mites is not limited to allergens. In this paper, genomic, transcriptomic and proteomic data on Dermatophagoides pteronyssinus (DP) was mined for information regarding the complete degradome of this house dust mite. D. pteronyssinus has more proteases than the closely related Acari, Dermatophagoides farinae (DF) and Sarcoptes scabiei (SS). The group of proteases in D. pteronyssinus is found to be more highly transcribed than the norm for this species. The distribution of protease types is dominated by the cysteine proteases like Der p 1 that account for about half of protease transcription by abundance, and Der p 1 in particular accounts for 22% of the total protease transcripts. In an analysis of protease stability, the group of allergens (Der p 1, Der p 3, Der p 6, and Der p 9) is found to be more stable than the mean. It is also statistically demonstrated that the protease allergens are simultaneously more highly expressed and more stable than the group of D. pteronyssinus proteases being examined, consistent with common assumptions about allergens in general. There are several significant non-allergen outliers from the normal group of proteases with high expression and high stability that should be examined for IgE binding. This paper compiles the first holistic picture of the D. pteronyssinus degradome to which humans may be exposed.


Assuntos
Antígenos de Dermatophagoides/análise , Proteínas de Artrópodes/análise , Cisteína Endopeptidases/análise , Dermatophagoides pteronyssinus/química , Serina Endopeptidases/análise , Alérgenos/análise , Alérgenos/genética , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Cisteína Endopeptidases/genética , Dermatophagoides pteronyssinus/genética , Estabilidade Enzimática , Filogenia , Alinhamento de Sequência , Serina Endopeptidases/genética
17.
Int J Mol Sci ; 18(5)2017 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-28531096

RESUMO

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.


Assuntos
Antígenos de Dermatophagoides/química , Proteínas de Artrópodes/química , Cisteína Endopeptidases/química , Precursores Enzimáticos/química , Sequência de Aminoácidos , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Cisteína Endopeptidases/genética , Dipeptídeos/química , Precursores Enzimáticos/genética , Concentração de Íons de Hidrogênio , Cinética , Mutação , Conformação Proteica , Desdobramento de Proteína , Proteólise , Tirosina/química
18.
Med Vet Entomol ; 31(3): 272-280, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28429373

RESUMO

Products manufactured from mass-cultured house dust mites, currently commercialized for the diagnosis and immunotherapy of allergy, are heterogeneous in terms of allergen composition and thus present concerns to regulatory authorities. The most abundant species, Dermatophagoides pteronyssinus (Trouessart) (Astigmata: Pyroglyphidae), produces 19 allergenic proteins. Many of these are putatively involved in mite digestive physiology and metabolism. This study aimed to evaluate the effects of mite-rearing media on allergen production. Mites were adapted to feed on culture media supplemented with proteins, lipids, carbohydrates or beard shavings, and collected to quantify major allergens (Der p 1 and 2) by immunodetection, transcription of allergen genes by real-time quantitative polymerase chain reaction, and allergen-related enzymatic activities. All culture media significantly affected the content of major allergens. Modification of macronutrients in the diet produced minor effects on the transcription of allergen genes, but significantly altered mite allergen-related activities. The most remarkable impacts were detected in mites feeding on beard shavings and were reflected in reductions in the content of major allergens, alterations in the transcription of nine allergen genes, and changes in eight allergen-related activities. These results demonstrate the importance of culture media to the quality and consistency of mite extracts used for pharmaceuticals, and highlight the need to further elucidate allergen production by mites in the laboratory and in domestic environments.


Assuntos
Alérgenos/metabolismo , Dermatophagoides pteronyssinus/fisiologia , Carboidratos da Dieta/administração & dosagem , Proteínas na Dieta/administração & dosagem , Lipídeos/administração & dosagem , Alérgenos/genética , Ração Animal/análise , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dermatophagoides pteronyssinus/enzimologia , Dermatophagoides pteronyssinus/genética , Dieta , Suplementos Nutricionais/análise , Expressão Gênica , Pele
19.
Pediatr Pulmonol ; 52(3): 282-292, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27434417

RESUMO

BACKGROUND: The house dust mite species Dermatophagoides farinae releases allergens that cause allergies and asthma worldwide. This study sought to clone and express the full-length cDNA encoding the group 9 allergen of D. farinae (Der f 9). METHODS: The published sequence of Der f 9 was used to design primers for RT-PCR and RACE to obtain the full-length cDNA encoding Der f 9. After removal of signal peptide sequence, Der f 9 was then sub-cloned into plasmid pET-28b (+), and the plasmid was transformed into Escherichia coli BL21 (DE3) cells for expression. The recombinant protein was purified by Nickel affinity chromatography, identified by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from children with asthma. Bioinformatics analyses were used to identify features of Der f 9. RESULTS: By RT-PCR, 3'-RACE, and 5'-RACE, the full-length sequence of Der f 9 was generated, which was confirmed by nucleotide sequencing. The mature Der f 9 was expressed successfully in E. coli, which was identified by SDS-PAGE. The recombinant allergen was purified by chromatography and confirmed by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF. Sera from 56.7% (17/30) of mite-allergic patients reacted with the purified recombinant Der f 9. CONCLUSIONS: The successful production of recombinant Der f 9 protein revealed the importance of Der f 9 in mite allergy, and provides a foundation for further study of this allergen in diagnosis and treatment of symptoms. Pediatr Pulmonol. 2017;52:282-292. © 2016 Wiley Periodicals, Inc.


Assuntos
Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Asma/imunologia , Animais , Sequência de Bases , Criança , Clonagem Molecular , Humanos , Imunoglobulina E/sangue , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Análise de Sequência de Proteína
20.
Mol Med Rep ; 14(5): 4816-4822, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27748932

RESUMO

Mimotope mapping enables the characterization of allergen epitopes for the development of diagnostic and therapeutic approaches. In the present study, a phage display peptide library was used for mimotope mapping based on the binding of antibodies against the recombinant group 5 allergen from the house dust mite Dermatophagoides farinae (Der f 5), an arthropod that causes indoor allergies worldwide. When three monoclonal anti­Der f 5 antibodies were used for biopanning, seven mimotopes were identified. Their common subsequence was '­­­[­A][­T]W[­S]H[HSFW][LM][PSKR] [TLV][AST]­[DP][­L]­'. When analyzed in combination with predicted discontinous epitopes, amino acids P2, K3, K4, H5, F11, F13, L14, R72, T77, L79, R84, T39, F40, P44, T45 and K46 were identified as key residues in conformational epitopes of Der f 5. Therefore, the seven mimotopes or modification of the key amino acids may facilitate the development of blocking antibodies or epitope­specific immunotherapies for mite allergy.


Assuntos
Antígenos de Dermatophagoides/imunologia , Técnicas de Visualização da Superfície Celular , Mapeamento de Epitopos , Epitopos/imunologia , Biblioteca de Peptídeos , Pyroglyphidae/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/isolamento & purificação , Mapeamento de Epitopos/métodos , Epitopos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
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