RESUMEN
Studies about thymic B cells are scarce in the literature, but it was suggested that they can exert modulatory and regulatory functions on the immune system. Thymic B cells can play some role in regulating the most frequent allergic background worldwide, the atopy induced by the mite Dermatophagoides pteronyssinus (Der p). Here, we aimed to evaluate if the polyclonal IgG repertoire produced by Der p-atopic individuals can influence the homing and cytokine profile of human thymic B derived from non-atopic children aged less than seven days. With this purpose, we produced polyclonal IgG formulations and cultivated human thymocytes in their presence. We also assessed IgG subclasses and the direct interaction of IgG with thymic B cell membranes. Our results could demonstrate that Der p-atopic IgG could not reduce the expression of α4ß7 homing molecule as observed in response to the other IgG formulations and could reduce the frequency of IFN-γ- and IL-9-producing thymic B cells compared to the mock condition. Der p-atopic IgG could also induce thymic IL-10-producing B cells compared to control conditions. The IgG derived from Der p-atopic individuals failed to diminish the population of IL-13-producing thymic B cells, unlike the reduction observed with other IgG formulations when compared to the mock condition. All IgG formulations had similar levels of IgG subclasses and directly interacted with thymic B cell membranes. Finally, we performed experiments using peripheral non-atopic B cells where IgG effects were not observed. In conclusion, our observation demonstrates that IgG induced in allergic individuals can modulate non-atopic thymic B cells, potentially generating thymic B cells prone to allergy development, which seems to not occur in mature B cells.
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Hipersensibilidad Inmediata , Hipersensibilidad , Animales , Niño , Humanos , Interleucina-10 , Dermatophagoides pteronyssinus , Interleucina-9 , Interferón gamma/metabolismo , Inmunoglobulina G , Fenotipo , Antígenos Dermatofagoides , AlérgenosRESUMEN
BACKGROUND: There is a lack of information on house dust mite (HDM) sensitization and phenotype distribution in patients with severe asthma (SA) living permanently at high-altitude (HA) in tropical regions, which may be different. OBJECTIVE: The aim of this study was to characterize adults with SA in a tropical high altitude city (2,640 m): Bogotá, Colombia. MATERIAL AND METHODS: This observational cross-sectional study included severe asthmatic outpatients (n = 129) referred to the ASMAIRE program of the Fundación Neumológica Colombiana in Bogotá (2,640 m). Clinical history, spirometry, total IgE, blood eosinophils, and skin prick test (SPT), including HDM allergens, were performed. Phenotype definitions: Allergic/atopic (AA): IgE ≥100 IU/mL and/or at least one positive SPT; eosinophilic (EOS): blood eosinophils ≥300 cells/µL; type 2-high: AA and/or EOS phenotype; type 2-low: non-AA/non-EOS phenotype (IgE <100 IU/mL, negative SPT, and blood eosinophils <300 cells/µL). RESULTS: A total of 129 adults with SA were included, 79.8% female. Phenotype distribution: AA: 61.2%; EOS: 37.2%; type 2-high: 72.1%; type 2-low: 27.9%. Among AA patients, HDM sensitization was present in 87% and 34.9% were non-eosinophilic. There was a significant overlap between the phenotypes. CONCLUSIONS: In contrast to non-tropical high-altitude regions, we found a high frequency of HDM sensitization in patients with AA phenotype living in a tropical high-altitude city. We also found a discrete lower frequency of EOS phenotype with no other significant differences in the phenotypic distribution compared to that described at low altitudes. We propose that tropical location may modify the effect of high altitude on HDM concentrations and allergenicity.
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Asma , Hipersensibilidad , Humanos , Adulto , Animales , Femenino , Masculino , Asma/epidemiología , Pyroglyphidae , Altitud , Inmunoglobulina E , Dermatophagoides pteronyssinus , Alérgenos , Pruebas Cutáneas , Antígenos Dermatofagoides , PolvoRESUMEN
Only few allergens derived from house dust mite (HDM) species have been evaluated in terms of their potential to induce allergic inflammation. In this study, we aimed to evaluate different aspects of the allergenicity and allergenic activity of Blo t 2, a Blomia tropicalis allergen. Blo t 2 was produced as a recombinant protein in Escherichia coli. Its allergenic activity was tested in humans by skin prick test and basophil activation assays, and in mice, by passive cutaneous anaphylaxis and a model of allergic airway inflammation. Sensitization rate to Blo t 2 (54.3%) was similar to that found to Blo t 21 (57.2%) and higher than to Der p 2 (37.5%). Most Blo t 2-sensitized patients showed a low intensity response (99.5%). Blo t 2 elicited CD203c upregulation and allergen induced skin inflammation. Additionally, immunized animals produced anti-Blo t 2 IgE antibodies and passive transfer of their serum to non-immunized animals induced skin inflammation after allergen exposure. Immunized animals developed bronchial hyperreactivity and a strong inflammatory lung reaction (eosinophils and neutrophils). These results confirm the allergenic activity of Blo t 2 and supports its clinical relevance.
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Alérgenos , Pyroglyphidae , Humanos , Ratones , Animales , Dermatophagoides pteronyssinus , Inmunoglobulina E , Inflamación , Antígenos DermatofagoidesRESUMEN
BACKGROUND: Allergen-specific immunotherapy (AIT) is the only clinical approach that can potentially cure some allergic diseases by inducing immunological tolerance. Dermatophagoides pteronyssinus is considered as the most important source of mite allergens worldwide, with high sensitization rates for the major allergens Der p 1, Der p 2 and Der p 23. The aim of this work is to generate a hypoallergenic hybrid molecule containing T-cell epitopes from these three major allergens. METHODS: The hybrid protein termed Der p 2231 containing T-cell epitopes was purified by affinity chromatography. The human IgE reactivity was verified by comparing those with the parental allergens. The hybrid was also characterized immunologically through an in vivo mice model. RESULTS: The hybrid rDer p 2231 stimulated in peripheral blood mononuclear cells (PBMCs) isolated from allergic patients with higher levels of IL- 2, IL-10, IL-15 and IFN-γ, as well as lower levels of IL-4, IL-5, IL-13, TNF-α and GM-CSF. The use of hybrid molecules as a therapeutic model in D. pteronyssinus allergic mice led to the reduction of IgE production and lower eosinophilic peroxidase activity in the airways. We found increased levels of IgG antibodies that blocked the IgE binding to the parental allergens in the serum of allergic patients. Furthermore, the stimulation of splenocytes from mice treated with rDer p 2231 induced higher levels of IL-10 and IFN-γ and decreased the secretion of IL-4 and IL-5, when compared with parental allergens and D. pteronyssinus extract. CONCLUSIONS: rDer p 2231 has the potential to be used in AIT in patients co-sensitized with D. pteronyssinus major allergens, once it was able to reduce IgE production, inducing allergen-specific blocking antibodies, restoring and balancing Th1/Th2 immune responses, and inducing regulatory T-cells.
Asunto(s)
Antígenos Dermatofagoides , Epítopos de Linfocito T , Hipersensibilidad , Animales , Humanos , Ratones , Alérgenos , Antígenos Dermatofagoides/inmunología , Antígenos Dermatofagoides/farmacología , Antígenos Dermatofagoides/uso terapéutico , Proteínas de Artrópodos , Dermatophagoides pteronyssinus , Epítopos de Linfocito T/química , Epítopos de Linfocito T/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Inmunoglobulina E , Interleucina-10 , Interleucina-4 , Interleucina-5 , Leucocitos Mononucleares , Pyroglyphidae , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inmunoterapia/métodosRESUMEN
Blomia tropicalis and Dermatophagoides pteronyssinus play an important role in triggering allergy. Glycycometus malaysiensis causes IgE reaction in sensitive people, but is rarely reported in domestic dust, because it is morphologically similar to B. tropicalis making the identification of these species difficult. The identification of mites is mostly based on morphology, a time-consuming and ambiguous approach. Herein, we describe a multiplex polymerase chain reaction (mPCR) assay based on ribosomal DNA capable to identify mixed cultures of B. tropicalis, D. pteronyssinus and G. malaysiensis, and/or to identify these species from environmental dust. For this, the internal transcribed spacer 2 (ITS2) regions, flanked by partial sequences of the 5.8S and 28S genes, were PCR-amplified, cloned and sequenced. The sequences obtained were aligned with co-specific sequences available in the GenBank database for primer design and phylogenetic studies. Three pairs of primers were chosen to compose the mPCR assay, which was used to verify the frequency of different mites in house dust samples (n = 20) from homes of Salvador, Brazil. Blomia tropicalis was the most frequent, found in 95% of the samples, followed by G. malaysiensis (70%) and D. pteronyssinus (60%). Besides reporting for the first time the occurrence of G. malaysiensis in Brazil, our results confirm the good resolution of the ITS2 region for mite identification. Furthermore, the mPCR assay proved to be a fast and reliable tool for identifying these mites in mixed cultures and could be applied in future epidemiological studies, and for quality control of mite extract production for general use.
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Dermatophagoides pteronyssinus , Ácaros , Animales , Antígenos Dermatofagoides , Brasil , Polvo , Humanos , Reacción en Cadena de la Polimerasa Multiplex , FilogeniaRESUMEN
Cryptococcosis (caused, for example, by Cryptococcus neoformans) and allergic asthma (caused, for example, by Dermatophagoides pteronyssinus) target the respiratory tract (the lung and bronchial epithelium). C. neoformans and D. pteronyssinus can coexist in the same indoor environment, and exposure to both can cause alterations in the local airway inflammatory milieu and exacerbation of airway inflammatory diseases. Here, we evaluated the effects of the association between C. neoformans and D. pteronyssinus in the modulation of airway inflammatory responses in an in vitro experimental model using human bronchial epithelial cells. BEAS-2B cells were cultivated and stimulated with D. pteronyssinus (10 µg/mL) and/or C. neoformans (MOI 100) for 24 h. No cytotoxic effect was observed in cells stimulated by C. neoformans and/or D. pteronyssinus. The production of IL-8, IL-6, and/or CCL2, but not IL-10, as well as the activation of NF-kB, STAT3, STAT6, and/or ERK1/2 were increased in cells stimulated by C. neoformans or D. pteronyssinus compared to controls. C. neoformans in association with D. pteronyssinus inhibited the CCL2ERK1/2 signaling pathway in cells treated with both pathogens compared to cells stimulated by D. pteronyssinus alone. In addition, their association induced an additive effect on the IL-6/STAT3 signaling pathway in cells compared to cells stimulated with D. pteronyssinus or C. neoformans only. D. pteronyssinus increased the internalization and growth of C. neoformans in BEAS-2B cells. D. pteronyssinus in association with C. neoformans promoted pro- and anti-inflammatory responses, which can modulate cryptococcal infection and asthmaticus status.
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Criptococosis , Cryptococcus neoformans , Animales , Antiinflamatorios/farmacología , Bronquios , Quimiocina CCL2/metabolismo , Criptococosis/metabolismo , Cryptococcus neoformans/metabolismo , Dermatophagoides pteronyssinus/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Humanos , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: Sensitization to house dust mites (HDMs) is frequent in patients with atopic dermatitis. OBJECTIVE: To investigate the efficacy of sublingual immunotherapy (SLIT) with Dermatophagoides pteronyssinus extract in patients with atopic dermatitis sensitized to HDM. METHODS: In this randomized, double-blind, placebo-controlled trial, we enrolled 91 patients 3 years or older, with SCORing Atopic Dermatitis (SCORAD) score greater than or equal to 15 and positive skin test result and/or IgE to D pteronyssinus. Patients were stratified according to age (<12 and ≥12 years) to receive HDM SLIT or placebo for 18 months. Primary outcome was a greater than or equal to 15-point decrease in SCORAD score. Secondary outcomes were decreases in SCORAD and objective SCORAD, Eczema Area and Severity Index, visual analog scale for symptoms, and pruritus scale scores; Investigator's Global Assessment 0/1; and decrease greater than or equal to 4 points in Dermatology Life Quality Index. Background therapy was maintained. RESULTS: A total of 66 patients completed the study (35 HDM SLIT, 31 placebo). After 18 months, 74.2% and 58% of patients in the HDM SLIT group and the placebo group, respectively, showed greater than or equal to 15-point decrease in SCORAD score (relative risk, 1.28; 95% CI, 0.89-1.83). Significant SCORAD score decreases from baseline of 55.6% and 34.5% in HDM SLIT and placebo groups (mean difference, 20.4; 95% CI, 3.89-37.3), significant objective SCORAD score decreases of 56.8% and 34.9% in HDM SLIT and placebo groups (mean difference, 21.3; 95% CI, 0.66-41.81), and more patients with Investigator's Global Assessment 0/1 in the HDM SLIT group as compared with the placebo group (14 of 35 vs 5 of 31; relative risk, 2.63; 95% CI, 1.09-6.39) were observed at 18 months. CONCLUSIONS: Our results suggest that HDM SLIT may be effective in HDM-sensitized patients as an add-on treatment for atopic dermatitis.
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Dermatitis Atópica , Eccema , Inmunoterapia Sublingual , Animales , Antígenos Dermatofagoides/uso terapéutico , Niño , Dermatitis Atópica/tratamiento farmacológico , Dermatophagoides pteronyssinus , Método Doble Ciego , Eccema/tratamiento farmacológico , Humanos , Pyroglyphidae , Inmunoterapia Sublingual/métodos , Resultado del TratamientoAsunto(s)
Alérgenos/efectos adversos , Alergia e Inmunología , Asma/terapia , Medicina de Precisión/métodos , Rinitis Alérgica/terapia , Alérgenos/metabolismo , Animales , Asma/sangre , Asma/genética , Asma/inmunología , Dermatophagoides pteronyssinus/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/metabolismo , Unión Proteica/inmunología , Rinitis Alérgica/sangre , Rinitis Alérgica/genética , Rinitis Alérgica/inmunología , Terminología como AsuntoRESUMEN
INTRODUCTION: Aqueous allergen injections, an effective and century-old technique, is considered a second-line approach in daily clinical practice. Inconveniences still surround conventional subcutaneous immunotherapy (SCIT) administration, such as a need for frequent injections, prolonged up-dosing schedules, elevated costs, and the unlikely possibility of a systemic reaction. The intradermal immunotherapy route (IDR) might favorably impact many of the aforementioned issues (Table 1). House dust mite (HDM) allergens are the main perennial sensitizers in the tropics, and as such, are solely employed in immunotherapy treatments. METHODS: We carried out a year-long real-life study in 25 perennial allergic rhinitis children, symptomatic on exposure to house dust, employing an intradermal low-dose allergen mix consisting of 50 ng of Dermatophagoides pteronyssinus/Dermatophagoides farinae and 120 ng of Blomia tropicalis, under a unique cost-wise protocol. Basal symptoms/signs and face Visual Analog Scale (fVAS) scores were recorded for 2 weeks and later compared with those registered throughout the 1-year treatment. Serum-specific IgG4 and IL-10 levels were employed in the assessment of the immune responses. RESULTS: Symptoms/signs and fVAS scores were significantly reduced from days 42 and 49, respectively, and remained so until treatment completion. Increases in specific IgG4's and IL-10 levels reflected significant immune responses. Injections were well tolerated and families reported improved health status (quality of life, QoL). CONCLUSIONS: A unique cost-effective immunotherapy alternative for deprived allergic communities in tropical settings is depicted; further research is needed.
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Alérgenos/administración & dosificación , Antígenos Dermatofagoides/administración & dosificación , Desensibilización Inmunológica/economía , Rinitis Alérgica Perenne/terapia , Adolescente , Alérgenos/inmunología , Animales , Antígenos Dermatofagoides/inmunología , Niño , Preescolar , Análisis Costo-Beneficio , Dermatophagoides farinae/inmunología , Dermatophagoides pteronyssinus/inmunología , Desensibilización Inmunológica/métodos , Países en Desarrollo , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inyecciones Intradérmicas , Interleucina-10/sangre , Interleucina-10/inmunología , Masculino , Calidad de Vida , Rinitis Alérgica Perenne/sangre , Rinitis Alérgica Perenne/diagnóstico , Rinitis Alérgica Perenne/inmunología , Índice de Severidad de la Enfermedad , Pruebas Cutáneas , Resultado del Tratamiento , Clima TropicalRESUMEN
Canine demodicosis is a common inflammatory parasitic skin disease caused by Demodex mites. House dust mites, such as Dermatophagoides spp., play an important role in the pathogenesis of canine atopic dermatitis (AD). The goal of this experimental work was to investigate whether demodectic dogs could be previously exposed/sensitized to house dust mites' antigens. First the prevalence of demodicosis in a southeastern region of Brazil was investigated by analyzing clinical files of dogs that were admitted to a Veterinary Hospital. Subsequently, the IgG responses to Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) and IgE to D.pteronyssinus (Dp) were evaluatedin two groups, AD or demodicosis dogs. Additionally, the major IgE-binding Dp proteins that are recognized by sera from dogs with demodicosis and AD were evaluated. A total of 2,599 clinical files were analyzed to identify the major parasitic skin diseases in dogs from this region, considering the age, sex and breed of the animals. The epidemiological study identified 111 animals with skin diseases; from these 20.7% presented demodicosis. Afterwards, serum samples were obtained from another groups of demodicosis, AD, and healthy dogs, and analyzed for Dp and Df-specific IgG, and IgE antibody levels, Dp IgG avidity by ELISA and IgE-binding Dp-specific proteins by immunoblot. IgG and IgE antibodies to Dp were detected in sera from additional groups of dogs with AD, demodicosis or healthy, with higher IgE levels to Dp in AD than demodectic or healthy dogs. IgG to Df was detected, despite with smaller levels compared to Dp in sera from demodectic dogs, and also in healthy dogs. Immunoblot showed IgE-binding to Dp proteins in sera of dogs with demodicosis and AD; with strong reactivity for the 72 and 116 kDa antigens detected by sera from demodicosis dogs. However, sera from healthy dogs >12 months old also presented reactivity to these bands. In conclusion, the detection of Dp-IgG and IgE antibodies in sera from demodectic dogs indicates previous exposure and sensitization to the house dust mite, respectively, more than cross-reactivity between demodex mites and Dp antigens detected by canine antibodies. Additionally, higher Dp-specific IgE levels were found in dogs with AD compared with those with demodicosis or healthy, suggesting that Dp-specific IgE could better discriminate dogs with AD from healthy ones or even those with demodicosis.
Demodicose canina é uma doença inflamatória comum da pele causada por ácaros do gênero Demodex. Ácaros da poeira doméstica como Dermatophagoides spp. desempenham papel importante na patogênese da dermatite atópica canina (DA). O objetivo desse trabalho experimental foi investigar se cães com demodicose poderiam ser previamente expostos/sensibilizados com antígenos de ácaros da poeira doméstica. A princípio, investigou-se a prevalência de demodicose em uma região sudeste do Brasil, analisando-se prontuários clínicos de cães admitidos em um Hospital Veterinário. Posteriormente, as respostas de IgG a Dermatophagoides pteronyssinus (Dp) e D. farinae (Df) e IgE a D. pteronyssinus (Dp) foram avaliadas em dois grupos, DA ou demodicose. Também foram avaliadas as principais proteínas Dp reconhecidas por anticorpo IgE presente em soros de cães com demodicose e DA. Um total de 2.599 prontuários clínicos foram analisados para identificar as principais doenças parasitárias da pele em cães dessa região, considerando a idade, sexo e raça dos animais. O estudo epidemiológico detectou 111 animais com doenças de pele e destes, 20,7% apresentavam demodicose. Posteriormente, amostras de soro foram obtidas de outros grupos de cães com demodicose, DA ou saudáveis, e analisadas quanto aos níveis de IgG e IgE específicos para Dp e Df, avidez de IgG a Dp por ELISA e proteínas específicas de Dp reconhecidas por IgE por immunoblot. Anticorpos IgG e IgE para Dp foram detectados em soros de grupos adicionais de cães com DA, demodicose ou saudáveis, com níveis mais altos de IgE para Dp na DA do que no soro de animais saudáveis. Níveis de IgG específicos para Df foram detectados, apesar serem menores em comparação com os detectados para Dp em soros de cães demodéticos, e também em cães saudáveis. A análise de immunoblot demonstrou detecção de IgE para proteinas de Dp em soros de cães com demodicose e DA; com forte reatividade para os antígenos de 72 e 116 kDa detectados por soros de cães com demodicose. No entanto, soros de cães saudáveis > 12 meses de idade também apresentaram reatividade a essas bandas. Em conclusão, a detecção de anticorpos Dp-IgG e IgE específicos em soros de cães demodéticos indica exposição prévia e sensibilização aos ácaros, respectivamente, mais do que reatividade cruzada entre ácaros Demodex e antígenos Dp detectados por anticorpos caninos. Além disso, níveis de Dp-IgE específicos mais elevados encontrados em cães com DA, sugerem que esses anticorpos poderiam discriminar melhor cães com DA daqueles saudáveis ou mesmo demodéticos.
Asunto(s)
Inmunoglobulina E , Inmunoglobulina G , Dermatophagoides pteronyssinus , PerrosRESUMEN
BACKGROUND: The prevalence of allergic diseases has increased worldwide. Recent studies have informed that the dysbiosis of some specific members of the human microbiota may enhance the allergic response of the respiratory tract. OBJECTIVE: To retrospectively explore the role of some microorganisms of the human microbiota on the skin reactivity and their effect on the chronicity of allergic respiratory diseases in humans. METHODS: A retrospective analysis of a 5-year database of patients with allergic respiratory tract disease. The frequency and magnitude of the reactivity to 38 different allergens was determined. RESULTS: Dermatophagoides pteronyssinus had the highest frequency of reactivity (93.7 %), followed by the bacterial allergen (a mixture of Staphylococcus aureus and Staphylococcus epidermidis) with a frequency of reactivity of 91.82 %; whereas Candida albicans had a frequency of reactivity of only 79.32 %. The frequency of reactivity to the pollen of native Mexican weeds was even lower ~79 %. CONCLUSION: The microorganisms of the microbiota that were analyzed in this study seem to have an influence on the development of respiratory allergic inflammation, associated with long-term colonization of the pharynx, nasal mucosa, and sinuses because of these microorganisms.
Antecedentes: La prevalencia de las enfermedades alérgicas ha aumentado en todo el mundo. En estudios recientes se ha informado que la disbiosis de algunos miembros específicos de la microbiota humana puede potenciar la respuesta alérgica de las vías respiratorias. Objetivo: Explorar retrospectivamente el papel de algunos microorganismos de la microbiota humana en la reactividad cutánea y su efecto sobre la cronicidad de las enfermedades alérgicas respiratorias en el humano. Métodos: Análisis retrospectivo de la base de datos de un periodo de cinco años de pacientes con enfermedad alérgica de las vías respiratorias. Se determinó la frecuencia y magnitud de la reactividad a 38 alérgenos diferentes. Resultados: La mayor frecuencia de reactividad la presentó Dermatophagoides pteronyssinus (93.7 %), al que le siguió una combinación bacteriana de Staphylococcus aureus-Staphylococcus epidermidis (91.82 %) y Candida albicans (79.32 %). La reactividad a alérgenos de polen de malezas nativas de México fue aun menor, aproximadamente de 79 %. Conclusión: Los microorganismos de la microbiota analizados en este estudio parecen tener una influencia en el desarrollo de la inflamación alérgica respiratoria, asociada a la colonización a largo plazo de la faringe, la mucosa nasal y los senos paranasales.
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Alérgenos/inmunología , Antígenos/inmunología , Microbiota/inmunología , Hipersensibilidad Respiratoria/inmunología , Sistema Respiratorio/inmunología , Animales , Antígenos Bacterianos/inmunología , Candida albicans/inmunología , Niño , Dermatophagoides pteronyssinus/inmunología , Femenino , Humanos , Masculino , Estudios Retrospectivos , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Adulto JovenRESUMEN
Introduction: Allergen proteins found in dust mite extracts, such as Dermatophagoides farinae (DF), Dermatophagoides pteronyssinus (DP) and Tyrophagus putrescentiae (TP), are relevant for scientific studies in the allergy and immunotherapy fields. The precipitation/concentration of protein extracts may favor the aggregation of the allergens in homogenates. Objective and method: This paper investigates the precipitation process by submitting crude mite extracts to compounds such as ammonium sulfate (NH4)2SO4, trichloroacetic acid (TCA) and acetone. Results: The best results were obtained by fractionation with (NH4)2SO4 at 80% (w/v) saturation (~0° C), observing the protein markings on the electrophoresis gel. Major allergens were identified by immunoblot at 25 kDa (cysteine protease) for Der f and Der p; and 25 kDa, 30 kDa (tropomyosin) and Try p 3, near 26 kDa. For this percentage the total protein contents were 12.83 mg mL-1 for Der f, 24.78 mg mL-1 for Der p and 27.35 mg mL-1 for Try. Conclusion: An advantage of precipitation with (NH4)2SO4 over precipitation with acetone was the possibility of gradually obtaining protein fractions, which does not happen when using the latter. The addition of 80% (v/v) acetone to the mite extracts favored total protein precipitation in the concentrations 16.42 mg mL-1, 28.47 mg mL-1 and 13.41 mg mL-1. The use of TCA in concentrations above 20% (w/v) forms peptides that are not retained in the gel under the established experimental conditions, and dilute solutions of this acid are more efficient.
Introdução: As proteínas alergênicas presentes nos extratos dos ácaros de poeira, tais como Dermatofagoides farinae (DF), Dermatofagoides pteronyssinus (DP) e Tyrophagus putrescentiae (TP) são relevantes para estudos científicos na área de alergias e aplicação em imunoterapias. A precipitação/concentração desses extratos proteicos pode favorecer a agregação de alérgenos nos homogenatos. Objetivo e método: O trabalho investiga o processo de precipitação, submetendo os extratos brutos de ácaros de poeira a compostos como sulfato de amônio (NH4)2SO4, ácido tricloroacético (ATC) e acetona. Resultados: Os melhores resultados foram obtidos por fracionamento com (NH4)2SO4 em 80% (m/v) de saturação (~ 0°C), observando as marcações proteicas no gel de eletroforese. Os alérgenos principais foram identificados por immunoblot em 25 kDa (cisteína protease) para Der f 1 e Der p 1; e 25 kDa, 33 kDa (tropomyosin), 11 kDa para Tyr. Para esse percentual, os teores de proteína total foram de 12.83 mg mL-1 para DF; 24,78 mg mL-1 para DP; e 27,35 mg mL-1 para TP. Conclusão: A vantagem da precipitação com (NH4)2SO4 frente à precipitação com acetona foi a possibilidade de gradativamente se obter frações proteicas, o que não acontece quando utilizado esse solvente. A adição de 80% (v/v) de acetona aos extratos de ácaros favoreceu a precipitação total de proteína nas concentrações 16,42 mg mL-1; 28,47 mg mL-1; e 13,41 mg mL-1. O uso de ATC em concentrações acima de 20% (m/v) forma peptídeos que não são retidos no gel nas condições experimentais estabelecidas, sendo eficiente soluções mais diluídas desse ácido.
Asunto(s)
Humanos , Tropomiosina , Dermatophagoides farinae , Dermatophagoides pteronyssinus , Proteasas de Cisteína , Sulfato de Amonio , Ácaros , Acetona , Péptido Hidrolasas , Solventes , Ácidos , Alérgenos , Proteínas , Polvo , HipersensibilidadRESUMEN
BACKGROUND: House dust mites are important allergen sources and some of these allergenic proteins may contain carbohydrate moieties, which are able to be isolated using lectins, as Concanavalin A (ConA). This study aimed to investigate allergenicity (IgE) and antigenicity (IgG1 and IgG4) of ConA-unbound and ConA-bound Dermatophagoides pteronyssinus (Dpt) crude extracts using sera of mite-allergic patients as well as inhibition capacity of antibody binding. MATERIAL AND METHODS: We obtained mannose-enriched and mannose-depleted fractions from Dpt by ConA affinity chromatography. Both ConA-bound and ConA-unbound fractions were evaluated by ELISA and Western Blotting for specific IgE, IgG1, and IgG4 reactivity with sera obtained from 95 mite-allergic patients (DP+) and 92 nonallergic (NA) subjects. Inhibition ELISA was used to assess cross-reactivity between Dpt extract and its fractions. RESULTS: Among the DP+ patients, no difference was found between ConA-unbound and ConA-bound fractions regarding the levels of specific IgE, IgG1, and IgG4. Nonallergic subjects had the same levels of specific IgG1 to both ConA-unbound and ConA-bound fractions, although for specific IgG4, values were higher for ConA-bound. A positive correlation was found among specific IgE, IgG1, and IgG4 levels when Dpt was compared to ConA-unbound and ConA-bound fractions. Recognition of crude Dpt by IgE, IgG1, and IgG4 was highly inhibited by ConA-unbound and ConA-bound fractions. Western Blotting revealed a broad spectrum of bands ranging from 14 to 116 kDa recognized by specific IgE and IgG4. However, IgG1 reached higher frequency values on high molecular weight polypeptides. CONCLUSION: ConA-unbound and ConA-bound fractions derived from D. pteronyssinus crude extract revealed important components involved in the IgE recognition in allergic patients as well as IgG1 and/or IgG4 in allergic and healthy subjects.
Asunto(s)
Alérgenos/inmunología , Dermatophagoides pteronyssinus/inmunología , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Adulto , Alérgenos/química , Animales , Concanavalina A/química , Concanavalina A/inmunología , Dermatophagoides pteronyssinus/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicosilación , Voluntarios Sanos , Humanos , Inmunoglobulina E/química , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Extractos Vegetales/química , Extractos Vegetales/inmunología , Pyroglyphidae/química , Pyroglyphidae/inmunologíaRESUMEN
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.
Asunto(s)
Alérgenos/uso terapéutico , Antígenos Dermatofagoides/uso terapéutico , Dermatophagoides pteronyssinus/inmunología , Hipersensibilidad/prevención & control , Alérgenos/genética , Alérgenos/inmunología , Animales , Antígenos Dermatofagoides/genética , Antígenos Dermatofagoides/inmunología , Dermatophagoides pteronyssinus/genética , Femenino , Humanos , Hipersensibilidad/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.
Asunto(s)
Alérgenos/inmunología , Anacardium/química , Inmunoglobulina E/inmunología , Polen/efectos adversos , Polen/química , Rinitis Alérgica Estacional/inducido químicamente , Adolescente , Adulto , Anciano , Alérgenos/química , Animales , Antígenos de Plantas/efectos adversos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/inmunología , Betula/metabolismo , Brasil , Proteínas Portadoras/análisis , Proteínas Portadoras/inmunología , Niño , Preescolar , Reacciones Cruzadas/inmunología , Dermatophagoides farinae , Dermatophagoides pteronyssinus , Femenino , Humanos , Masculino , Proteínas de Plantas/efectos adversos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Polen/genética , Proteómica , Rinitis Alérgica Estacional/inmunología , Pruebas CutáneasRESUMEN
BACKGROUND: The skin prick test is recognized as the gold standard for the specific diagnosis of allergy. OBJECTIVE: To determine the sensitivity and specificity of the epicutaneous patch test that has been modified for the diagnosis of respiratory allergy to Dermatophagoides pteronyssinus. METHODS: An observational, interpretative, prolective, and transversal study. The included patients were between 6 and 49 years old, with symptoms of a respiratory allergy, and a skin test was performed on them by using the European standardized prick test and a modified epicutaneous patch test with the variable of the scarification that occurred before the antigen was applied, and its homogenization with a wooden applicator, which occurred after the antigen was applied. The levels of sensitivity, specificity, and prevalence influence and positive and negative predictive values were calculated. RESULTS: 104 patients were included, of which 72 (69.2%) were women. The age category of the patients was 24 ± 11.3 years of age. 78.9% of the patients were diagnosed with allergic rhinitis, 1.9% were diagnosed with asthma, and 19.2% were diagnosed with both rhinitis and asthma. The prevalence of the respiratory allergy to Dermatophagoides pteronyssinus was of 62.5%; 94.23% with a correct diagnosis. The sensitivity and specificity of the test was of 93.85% and 94.87%; the positive and negative predictive values were of 96.83% and 90.24%, and the positive and negative likelihood ratios were of 18.30 and 0.06. CONCLUSIONS: The modified epicutaneous patch test was highly sensitive and specific; it decreased the risk of allergen carryover and it lowered the cost of the material that was used.
Antecedentes: La prueba por punción cutánea es el estándar de oro en el diagnóstico específico de alergia. Objetivo: Determinar sensibilidad y especificidad de la prueba epicutánea modificada para el diagnóstico de alergia respiratoria a Dermatophagoides pteronyssinus. Métodos: Estudio observacional, interpretativo, prolectivo y transversal. Se incluyeron pacientes de seis a 49 años de edad, con cuadro clínico de alergia respiratoria, en quienes se realizó punción cutánea con técnica europea y prueba epicutánea con la variante de escarificación previa a la aplicación del antígeno y posterior homogenización del mismo con aplicador de madera. Se calculó sensibilidad, especificidad, influencia de la prevalencia, valores predictivos positivo y negativo. Resultados: Se incluyeron 104 pacientes, 72 (69.2 %) del sexo femenino. La edad fue de 24 ± 11.3 años, 78.9 % tuvo diagnóstico de rinitis alérgica, 1.9 % de asma y 19.2 % de rinitis y asma. La prevalencia de alergia respiratoria a Dermatophagoides pteronyssinus fue de 62.5 %; 94.23 % estaba correctamente diagnosticado. La sensibilidad y especificidad de la prueba fue de 93.85 y 94.87 %, los valores predictivos positivo y negativo de 96.83 y 90.24 % y los cocientes de probabilidades positivo y negativo de 18.30 y 0.91. Conclusiones: La prueba epicutánea modificada fue altamente sensible y específica, disminuyó el riesgo de contaminación por arrastre de alérgenos y los costos en el material utilizado.
Asunto(s)
Asma/diagnóstico , Asma/inmunología , Dermatophagoides pteronyssinus/inmunología , Pruebas del Parche/métodos , Rinitis Alérgica/diagnóstico , Rinitis Alérgica/inmunología , Adolescente , Adulto , Animales , Niño , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Objective: The mite Cheyletus malaccensis is cited in the literature as a predator of other mite species. Little is known about its protein composition, and few studies have evaluated its ability to trigger atopic respiratory allergic reactions. The present study aims to investigate the protein profile fingerprint present in Cheyletus malaccensis extract and to evaluate its immunologic reactivity in the presence of specific immunoglobulins (IgE) from the serum of individuals diagnosed with allergy to the mites Dermatophagoides farinae, Dermatophagoides pteronyssinus and Blomia tropicalis. These three species carry proteins responsible for the most cases of atopic respiratory allergies, hence the interest in comparing them to Cheyletus malaccensis. Methods: Samples of aspirated dust containing Cheyletus malaccensis were collected from households in the city of Rio de Janeiro, Brazil. From the collected mass of this mite, extracts were prepared for analysis. Proteins present in the extracts were identified by electrophoresis under denaturing conditions. Results: Proteins with a molecular mass of 24 kDa, 26 kDa, 12 kDa, 45 kDa and 70 kDa were visualized. The immunoblotting assay showed positive cross-reactivity for proteins of molecular mass ranging from 20 kDa to 45 kDa. These results indicate that specific links were established between IgE present in the serum of individuals allergic to the comparator mite and proteins from Cheyletus malaccensis. Conclusions: These findings are relevant for their potential clinical and immunotherapeutic applications, as well as information base for further studies.
Objetivo: O ácaro Cheyletus malaccensis é referido na literatura como um predador de outras espécies de ácaro. Pouco se sabe sobre sua composição proteica, e poucos estudos avaliaram sua habilidade de desencadear reações alérgicas respiratórias atópicas. O objetivo do presente estudo é investigar a impressão digital do perfil proteico presente em um extrato de Cheyletus malaccensis e avaliar sua reatividade imunológica na presença de imunoglobulinas (IgE) específicas do soro de indivíduos diagnosticados com alergia aos ácaros Dermatophagoides farinae, Dermatophagoides pteronyssinus e Blomia tropicalis. Essas três espécies carregam proteínas responsáveis pela maioria dos casos de alergias respiratórias atópicas, o que justifica o interesse em compará-las ao Cheyletus malaccensis. Métodos: Amostras de poeira aspirada contendo Cheyletus malaccensis foram coletadas de domicílios na cidade do Rio de Janeiro, no Brasil. A partir da massa coletada desse ácaro, extratos foram preparados para análise. As proteínas presentes nos extratos foram identificadas por eletroforese sob condições desnaturantes. Resultados: Proteínas com massa molecular de 24 kDa, 26 kDa, 12 kDa, 45 kDa e 70 kDa foram visualizadas. O ensaio imunoenzimático mostrou reatividade cruzada positiva para proteínas de massa molecular variando de 20 kDa a 45 kDa. Esses resultados indicam que ligações específicas foram estabelecidas entre a IgE presente no soro de indivíduos alérgicos ao ácaro usado como comparador e proteínas de Cheyletus malaccensis. Conclusões: Os achados são relevantes por seu potencial clínico e aplicações imunoterapêuticas, bem como sua base de informações para futuros estudos.
Asunto(s)
Humanos , Proteínas , Dermatophagoides farinae , Dermatophagoides pteronyssinus , Hipersensibilidad , Inmunoglobulina E , Titulación a Punto Final de Prueba Cutánea , Electroforesis , Métodos , ÁcarosRESUMEN
Objetivos: Comparar os resultados obtidos com agulha e Multi-Test II® em testes cutâneos por puntura com diferentes concentrações de histamina e de extrato de Dermatophagoides pteronyssinus e a dor relatada em cada teste. Métodos: Estudo experimental, realizado no complexo Hospital de Clínicas em Curitiba, Paraná. Foram incluídas no estudo 104 crianças com idade entre 6 e 15 anos, com diagnóstico de asma e/ou rinite e/ou dermatite atópica e teste cutâneo alérgico positivo para Dermatophagoides pteronyssinus. Foram realizados testes com agulha hipodérmica descartável BD Precision Glide® 13 x 0,3 e com dispositivo Multi-test II® com histamina 10 mg/mL e 1 mg/ mL, Dermatophagoides pteronyssinus 5000 PNU/mL e 10000 PNU/mL e solução salina. Avaliação da dor foi obtida após cada teste pela escala de faces de dor de Wong-Baker. Resultados: A sensibilidade do teste cutâneo alérgico para os dois dispositivos foi 100% nas concentrações de histamina 10 mg/mL. Com histamina 1 mg/mL o Multi-test II® apresentou maior valor de sensibilidade (S = 86,5%) que a agulha (S = 56,7%). Alto nível de concordância entre os dois dispositivos foi observada com extrato de Dermatophagoides pteronyssinus na concentração de 10000 PNU/mL. Com a concentração de 5000 PNU/mL, o nível de concordância entre os testes foi 69,1% (Kappa = 0,2). A dor foi relatada por 65 (62,5%) crianças com Multi-Test II®, e 48 (46,2%) com agulha (p = 0,01). Conclusão: Houve alta sensibilidade para os dispositivos utilizados. Houve diferenças entre os tamanhos das pápulas nos testes cutâneos alérgicos com os dois dispositivos, porém resultados falso-positivos foram pouco observados. Ambos os dispositivos foram bem tolerados pelas crianças.
Objectives: To compare results obtained with needle and with Multi-Test II® in skin prick tests with different concentrations of histamine and Dermatophagoides pteronyssinus, and the pain reported by patients with each device. Methods: This experimental study was conducted at Hospital de Clínicas, Curitiba, Brazil. A total of 104 children were included, aged 6 to 15 years and diagnosed with asthma and/or rhinitis and/or atopic dermatitis and showing a positive allergic skin test for Dermatophagoides pteronyssinus. Tests were performed using 13 x 0.3 BD Precision Glide® disposable hypodermic needles and also Multi Test II® with histamine 10 mg/mL and 1 mg/mL, Dermatophagoides pteronyssinus extract 5000 PNU/mL and 10000 PNU/mL, and saline solution. Pain was evaluated after each test using the Wong-Baker FACES Pain Rating Scale. Results: Allergic skin test sensitivity was 100% for the two devices with histamine 10 mg/ mL. With histamine 1 mg/mL, the Multi-test II® showed higher sensitivity (S = 86.5%) than the needle (S = 56.7%). A high level of concordance between the two devices was observed with Dermatophagoides pteronyssinus extract at the 10000 PNU/ mL concentration; with 5000 PNU/mL, the level of concordance was 69.1% (Kappa = 0.2). Pain was reported by 65 (62.5%) children after the use of Multi-Test II® and by 48 (46.2%) after the use of needles (p = 0.01). Conclusion: High sensitivity rates were observed for both single or multiple devices. There were differences in wheal sizes between the two devices, but few falsepositive results were found. Both devices were well tolerated by children.
Asunto(s)
Humanos , Niño , Adolescente , Dolor , Asma , Dimensión del Dolor , Pruebas Cutáneas , Rinitis , Sensibilidad y Especificidad , Dermatophagoides pteronyssinus , Dermatitis Atópica , Pacientes , Piel , Diagnóstico , Equipos y SuministrosRESUMEN
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 22.8â¯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.