RESUMO
Mites are among the major sources of domestic and occupational allergens worldwide, and continuous exposure to these allergens leads to chronic airway inflammation. One of the most allergenic species is the storage mite Tyrophagus putrescentiae (Schrank). Protein extracts are produced from this mite for tests that help the clinical diagnosis (via prick test), treatment, and monitoring of disease progression in patients who had positive results for allergic reactions. Therefore, the aim of the present study was to evaluate the cell viability of RAW 264.7 and L929 cells when exposed to in-house raw protein extracts of T. putrescentiae compared to a commercial product, as well as quantify TNF-α secretion by RAW 264.7. Additionally, this study quantified the effect of these extracts in IgE secretion in total blood of people affected by this mite. The study found similarity between the in-house extract and the commercial extract as they had equivalent TNF-α secretion. Additionally, viabilities of RAW 264.7 and L929 exposed to the in-house extract were compatible with viabilities of cells exposed to the commercial extract, with no cytotoxicity at the concentrations tested. Results corroborated the hypothesis that the extract produced in-house would be equivalent to the commercial extract in allergic patients when the IgE was quantified. This study is the first to show the cytotoxicity of T. putrescentiae extracts, and to provide a quantitative analysis of TNF-α and IgE.
Assuntos
Acaridae , Hipersensibilidade , Ácaros , Humanos , Animais , Camundongos , Fator de Necrose Tumoral alfa , Imunoglobulina E , Reações Cruzadas , Alérgenos , Ácaros/metabolismoRESUMO
Successful research in the wide-ranging field of allergy is usually achieved by definition not only of physicochemical and immunological properties of natural, but also recombinant allergens. Blomia tropicalis mite is a well-known source for various groups of hypersensitivity-causing proteins. The goal of the present work was to produce, purify and characterise by in silico, biochemical and immunological methods the recombinant group-12 allergen of B. tropicalis. The recombinant Blo t 12 aggregation capacity as well as the affinity to antibodies from BALB/c immunised mice and B. tropicalis-sensitised human donors were investigated through in silico analyses, dynamic light scattering, SDS-PAGE, ELISA and Western blot. The presence of Blo t 12 within B. tropicalis extracts was also determined by ELISA and Western blot. High concentrations of dimeric rBlo t 12 were detected through SDS-PAGE next to other aggregates and the results were confirmed by data from DLS and Western blot. The YITVM peptide was predicted to be the most aggregation-prone region. The IgE-reactivity of rBlo t 12 was not completely abolished by aggregate formation but it was significantly decreased compared to rBlo t 5, or B. tropicalis extracts. Natural Blo t 12 may naturally dimerises, but it was detected in non-delipidified B. tropicalis extracts in low amounts. Given that this allergen may be a specific marker for B. tropicalis allergy, the recombinant Blo t 12 herein obtained is characterised as a mid-tier allergen in Brazilian atopic patients and may be useful for the improvement in precision allergy molecular diagnostic applications.
Assuntos
Alérgenos/isolamento & purificação , Ácaros/metabolismo , Alérgenos/genética , Alérgenos/imunologia , Animais , Escherichia coli/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas RecombinantesRESUMO
Cross-reactivity between allergens and human proteins could have a clinical impact in allergic diseases. Blo t 13 is an allergen from the mite Blomia tropicalis, which belongs to the fatty acid binding protein (FABP) family and has structural homology with human FABPs. This work aimed to map B cell epitopes on Blo t 13 and to identify epitopes involved in cross-reactivity with human heart FABP (FABP3) and adipocyte FABP (FABP4). Sera from 25 patients with house dust mite (HDM) allergy that were sensitized to Blo t 13 were used for testing the reactivity of immunoglobulin E (IgE) and IgG to FABP. The epitope mapping of Blo t 13 was performed using overlapping peptides, and cross-reactivity between Blo t 13 and human FABP was analyzed using human sera and anti-Blo t 13 monoclonal antibodies. IgE antibodies to all FABPs were detected in 14/25 serum samples, and IgG was detected in 25/25 serum samples. The cross-reactivity of Blo t 13 was 42% with FABP3 and 48% with FABP4. Two IgE-binding regions were identified in Blo t 13; one between residues 54 and 72 (the main cross-reacting region) and another between residues 111 to 129. Our results suggest that exposure to the Blo t 13 allergen could induce an auto-reactive response to endogenous FABP in allergic patients sensitized to Blo t 13.
Assuntos
Alérgenos/metabolismo , Epitopos de Linfócito B/imunologia , Proteína 3 Ligante de Ácido Graxo/imunologia , Proteínas de Ligação a Ácido Graxo/imunologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Adipócitos/metabolismo , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Mapeamento de Epitopos , Proteína 3 Ligante de Ácido Graxo/química , Proteína 3 Ligante de Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/patologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Masculino , Ácaros/metabolismo , Miocárdio/metabolismo , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
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.
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/metabolismoRESUMO
Frogs of the genus Phyllobates from Colombia are known to contain the highly toxic alkaloid batrachotoxin, but species from Central America exhibit only very low levels or are entirely free of this toxin. In the present study alcohol extracts from 101 specimens of Phyllobates lugubris and Phyllobates vittatus and 21 of three sympatric species (Dendrobates pumilio, Dendrobates auratus, Dendrobates granuliferus) from Costa Rica were analyzed by gas chromatography-mass spectrometry. Whereas the extracts of the Dendrobates species exhibited typical profiles of toxic alkaloids, those of the two Phyllobates species contained low levels of few alkaloids only, batrachotoxin was not detected. Although the feeding pattern of the Dendrobates and Phyllobates species are similar as revealed by examination of their stomach content (mainly ants and mites), the Phyllobates species are poorly sequestering alkaloids from their food source in contrast to the Dendrobates frogs.
Assuntos
Alcaloides/química , Anuros/classificação , Anuros/metabolismo , Animais , Formigas/metabolismo , Batraquiotoxinas/química , Costa Rica , Cromatografia Gasosa-Espectrometria de Massas , Ácaros/metabolismo , Pele/metabolismoRESUMO
La anafilaxia oral por ácaros es un nuevo síndrome caracterizado por síntomas alérgicos graves que se presentan en individuos alérgicos rápidamente después de la ingestion de alimentos confeccionados con harinas de trigo contaminadas con ácaros. Este síndrome más común en ambientes tropicales, es desencadenado más frecuentemente por panquecas y por ello ha sido designado "el síndrome de las panquecas". Se postula que los alergenos responsables son alergenos termorresistentes ya que los alimentos cocinados son capaces de inducir los síntomas. Una variedad del síndrome puede presentarse durante el ejercicio físico (anafilaxia inducida por ejercicio asociada a la ingestión de polvo con ácaros). Se recomienda conservar las harinas de trigo en el refrigerador para prevenir la proliferación de los ácaros y la aparición del cuadro clínico.
Oral mite anaphylaxis is a new syndrome characterized by severe allergic symptoms occurring in allergic patients immediately after the intake of foods made with mite-contaminated wheat flour. This syndrome, which is more common in tropical environments, is triggered more aften by pancakes and for that reason it has been designated "the pancake syndrome". Since cooked foods are able to induce the symptoms, it has been suggested that thermoresistant allergens are involved. A variety of this syndrome can occur during physical exercise (dust mite-ingestion associated exercise induced anaphylaxis). In order to prevent mite proliferation and the production of the clinical picture it is recommended to store wheat flours in the refrigerator.
Assuntos
Humanos , Masculino , Adolescente , Feminino , Criança , Ácaros/metabolismo , Anafilaxia/complicações , Anafilaxia/etiologia , Hipersensibilidade/etiologia , Alérgenos/análise , Contaminação de Alimentos , Triticum/efeitos adversosRESUMO
BACKGROUND: Ascaris lumbricoides induces a Th2 response and specific IgE synthesis in humans. This confers antiparasite immunity but could modify the natural history of allergic diseases in the tropics, justifying the study of its allergenic composition. We analyzed the allergenic properties of Ascaris tropomyosin and the frequency of sensitization in subjects exposed to the parasite. METHODS: cDNA was obtained by reverse transcription PCR, cloned into pQE30-UA and purified as a 6× His-tagged protein. Equivalence with its natural counterpart was analyzed by cross-inhibition and liquid chromatography-tandem mass spectrometry. Specific IgE was measured by ELISA in 175 asthmatics and 170 nonasthmatics naturally exposed to the parasite and sensitized to the Ascaris extract. RESULTS: The cDNA encoded 287 residues with high sequence identity with other invertebrate tropomyosins. The 40-kDa protein was recognized by human serum and affinity-purified anti-rBlo t 10 IgE. Specific IgE to tropomyosin could represent approximately 50% of the total IgE response to the extract. Ascaris tropomyosin induced wheal and flare in skin prick tests and histamine release from basophils. Although the prevalence of IgE to Ascaris tropomyosin was higher in asthmatic patients, logistic regression analysis suggested that this result was biased by sensitization to mites. CONCLUSIONS: A. lumbricoides tropomyosin (Asc l 3) is a new allergen that binds specific IgE, induces mediator release from effector cells and is cross-reactive to mite tropomyosins. IgE reactivity to this allergen is very frequent in both asthmatic and normal subjects sensitized to Ascaris extract. The potential role of Ascaris tropomyosin in asthma pathogenesis in tropical regions should be further investigated.
Assuntos
Alérgenos/imunologia , Ascaris lumbricoides/imunologia , Asma/imunologia , Imunoglobulina E/sangue , Tropomiosina/imunologia , Adolescente , Adulto , Alérgenos/genética , Sequência de Aminoácidos , Animais , Ascaris lumbricoides/metabolismo , Criança , Colômbia , Reações Cruzadas , Feminino , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Ácaros/imunologia , Ácaros/metabolismo , Dados de Sequência Molecular , Análise de Sequência de DNA , Testes Cutâneos , Clima Tropical , Tropomiosina/genética , Adulto JovemRESUMO
A total of 232 alkaloids, representing 21 structural classes were detected in skin extracts from the dendrobatid poison frog Oophaga pumilio, collected from 53 different populations from over 30 years of research. The highly toxic pumiliotoxins and allopumiliotoxins, along with 5,8-disubstitiuted and 5,6,8-trisubstituted indolizidines, all of which are proposed to be of dietary mite origin, were common constituents in most extracts. One decahydroquinoline (DHQ), previously shown be of ant origin, occurred in many extracts often as a major alkaloid, while other DHQs occurred rather infrequently. Histrionicotoxins, thought to be of ant origin, did not appear to possess a specific pattern of occurrence among the populations, but when present, were usually found as major components. Certain 3,5-disubstituted pyrrolizidines and indolizidines, known to be of ant origin, did occur in extracts, but infrequently. Alkaloid composition differed with regard to geographic location of frog populations, and for populations that were sampled two or more times during the 30-year period significant changes in alkaloid profiles sometimes occurred. The results of this study indicate that chemical defense in a dendrobatid poison frog is dependent on geographic location and habitat type, which presumably controls the abundance and nature of alkaloid-containing arthropods.
Assuntos
Alcaloides/química , Venenos de Anfíbios/química , Anuros/fisiologia , Ecossistema , Venenos/química , Animais , Formigas/metabolismo , Cromatografia Gasosa , Costa Rica , Geografia , Ácaros/metabolismo , Estrutura Molecular , Panamá , Pele/química , Fatores de Tempo , Extratos de Tecidos/químicaRESUMO
Alkaloids in the skin glands of poison frogs serve as a chemical defense against predation, and almost all of these alkaloids appear to be sequestered from dietary arthropods. Certain alkaloid-containing ants have been considered the primary dietary source, but dietary sources for the majority of alkaloids remain unknown. Herein we report the presence of approximately 80 alkaloids from extracts of oribatid mites collected throughout Costa Rica and Panama, which represent 11 of the approximately 24 structural classes of alkaloids known in poison frogs. Forty-one of these alkaloids also occur in the dendrobatid poison frog, Oophaga pumilio, which co-occurs with the collected mites. These shared alkaloids include twenty-five 5,8-disubstituted or 5,6,8-trisubstituted indolizidines; one 1,4-disubstituted quinolizidine; three pumiliotoxins; and one homopumiliotoxin. All but the last of these alkaloid classes occur widely in poison frogs. In addition, nearly 40 alkaloids of unknown structure were detected in mites; none of these alkaloids have been identified in frog extracts. Two of these alkaloids are homopumiliotoxins, five appear to be izidines, four appear to be tricyclics, and six are related in structure to poison frog alkaloids that are currently unclassified as to structure. Mites are common in the diet of O. pumilio, as well as in the diets of other poison frogs. The results of this study indicate that mites are a significant arthropod repository of a variety of alkaloids and represent a major dietary source of alkaloids in poison frogs.
Assuntos
Alcaloides/metabolismo , Ácaros/metabolismo , Ranidae/metabolismo , Alcaloides/química , Alcaloides/classificação , Animais , Costa Rica , Estrutura MolecularRESUMO
Reproduction and population growth of Varroa destructor was studied in ten naturally infested, Africanized honey bee (AHB) (Apis mellifera) colonies in Yucatan, Mexico. Between February 1997 and January 1998 monthly records of the amount of pollen, honey, sealed worker and drone brood were recorded. In addition, mite infestation levels of adult bees and worker brood and the fecundity of the mites reproducing in worker cells were determined. The mean number of sealed worker brood cells (10,070 +/- 1,790) remained fairly constant over the experimental period in each colony. However, the presence and amount of sealed drone brood was very variable. One colony had drone brood for 10 months and another for only 1 month. Both the mean infestation level of worker brood (18.1 +/- 8.4%) and adult bees (3.5 +/- 1.3%) remained fairly constant over the study period and did not increase rapidly as is normally observed in European honey bees. In fact, the estimated mean number of mites fell from 3,500 in February 1997 to 2,380 in January 1998. In May 2000 the mean mite population in the study colonies was still only 1,821 mites. The fertility level of mites in this study was much higher (83-96%) than in AHB in Brazil (25-57%). and similar to that found in EHB (76-94%). Mite fertility remained high throughout the entire study and was not influenced by the amount of pollen, honey or worker brood in the colonies.
Assuntos
Abelhas/parasitologia , Ácaros/crescimento & desenvolvimento , Animais , Feminino , Mel , Masculino , México , Ácaros/metabolismo , Pólen/metabolismo , Estações do Ano , Clima TropicalRESUMO
Any biocontrol agent used against stored grain insects will have to interact with insecticides used as grain protectants due to their heavy use especially in tropical areas. Therefore, the interaction between the pyrethroid insecticides bifenthrin and deltamethrin and Acarophenax lacunatus (Cross & Krantz) (Prostigmata: Acarophenacidae), an egg parasite of the stored cereal pest Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), was assessed in a range of mite densities and insecticide doses for each compound. Despite the mite species presence on all insecticide doses, the lowest instantaneous rate of increase of A. lacanatus was recorded with the highest doses of insecticides. The presence of the biological control agent at all doses of both insecticides suggests its tolerance to these compounds. A. lacunatus showed higher parasitism rate with bifenthrin than with deltamethrin indicating a higher selectivity of the first compound to the biocontrol agent.
Assuntos
Besouros/crescimento & desenvolvimento , Inseticidas/metabolismo , Ácaros/crescimento & desenvolvimento , Controle Biológico de Vetores/métodos , Triticum/parasitologia , Animais , Besouros/parasitologia , Feminino , Ácaros/metabolismo , Nitrilas , Piretrinas/metabolismo , Clima TropicalRESUMO
The objective of this study was to collect baseline information for implementing an acaricide resistance management program of Brevipalpus phoenicis (Geijskes) to hexythiazox in Brazilian citrus groves. The egg susceptibility of B. phoenicis to hexythiazox was measured by a direct contact bioassay. The estimated LC50 for the S strain was 0.89 mg hexythiazox L(-1) of water (95% FL 0.75-1.03). After hexythiazox selection of a field-collected population associated with intense hexythiazox use, a resistance ratio greater than 10,000-fold was detected. Results from a survey revealed a great variability in the frequency of resistance in populations of B. phoenicis collected from citrus groves located in the State of São Paulo. No relationship was observed between the intensity of hexythiazox use and the frequency of resistance. Studies on dynamics of resistance showed that the resistance of B. phoenicis to hexythiazox is stable under laboratory conditions. Therefore, there is an urgent need to implement resistance management of B. phoenicis to hexythiazox in order to prolong its effective use in Brazilian citrus groves.