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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. [ABSTRACT FROM AUTHOR]

The expression of allergen genes in house dust mites is influenced by temperature and relative humidity, but little is known of the impacts of other environmental factors that may alter the repertoire of allergens released by mites in home microhabitats. Bioassays were conducted in concave microscope slides in combination with real-time quantitative polymerase chain reaction (RT-qPCR) to analyse gene expression of 17 allergens of Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) exposed to three chemical stressors that can be present in domestic environments. Short-term exposure (5-12 days) to diesel exhaust particles (DEPs) (1 µg/cm2), bacterial lipopolysaccharide endotoxin (0.1 µg/cm2) and benzyl benzoate (3.2 µg/cm2), at concentrations exceeding those expected in homes, had no significant effect on allergen transcription. A significant increase in the transcription of allergens Der p 3, Der p 8 and Der p 21 was observed only after exposing mites to a higher concentration of DEPs (10 µg/cm2) over a whole generation. In combination, the present results suggest that the analysed factors have low impact on allergen production. The methodology described here offers a sound and rapid approach to the broad-spectrum study of factors affecting allergen-related mite physiology, and allows the simultaneous screening of different factors in a relatively short period with consideration of the full spectrum of allergen genes. [ABSTRACT FROM AUTHOR]

House dust mites are a major source of allergy worldwide. While diagnosis and treatment based on mite extracts have remarkably advanced, little information exists on the expression of allergens in mites. We have studied gene expression of eight Dermatophagoides pteronyssinus ( Trouessart) ( Acari: Pyroglyphidae) allergens ( Der p 1, 2, 3, 4, 5, 7, 10 and 21). All allergens showed higher transcription in nymphs compared with larvae or adults, with the only exception of Der p 10. The transcription of Der p 4 and Der p 10, together with the transcription and protein ratios Der p 1 to Der p 2, were higher in males than in females. One-week exposure of mite cultures to 16 or 35 °C (versus 24 °C) or low RH (44% versus 76%) significantly influenced the allergen gene transcription profile. Our results demonstrate that allergen expression is quantitatively and/or qualitatively influenced by mite development and sex, as well as by the environment. We suggest that monitoring allergen gene expression may be a useful tool to assist the optimization of mite cultures in the production of standardized allergenic extracts for clinical use. [ABSTRACT FROM AUTHOR]

. The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA ( rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers ( ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction ( PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species. [ABSTRACT FROM AUTHOR]

Trypsin, chymotrypsin, cathepsins B and D, aminopeptidase and carboxypeptidases A and B were detected in body extracts of the storage miteAcarus farris(Oudemans) (Astigmata: Acaridae). Faeces-enriched medium exhibited higher (10–50-fold) specific protease activity rates than those measured with mite body extracts for trypsin, chymotrypsin and carboxypeptidases A and B, suggesting that they are involved in mite digestion. However, the activity of cathepsin B was only three-fold higher in faecal than in body extracts, indicating that its presence in the lumen of the digestive tract is low compared to that of serine proteases. The activity of aminopeptidases was higher in mite bodies, indicating that they might be membrane bound. Cathepsin D activity was only detected in body extracts, indicating that this enzyme is not a digestive protease in this species. Zymograms resolved three major bands of gelatinolytic activity, but at least one protease form was only present in body extracts. Protease inhibitors of different specificity were testedin vivoto establish their potential as control agents. The development ofA. farriswas significantly retarded when the immature stages were fed on artificial diet containing inhibitors of serine and cysteine proteases and aminopeptidases, whereas no such effect was found with inhibitors of aspartyl proteases and carboxypeptidases. Interestingly, the most significant effects onA. farrisoccurred when a combination of inhibitors targeting different enzyme classes was supplied mixed in the diet, suggesting a synergistic toxicity. Several plant lectins were also tested, but only wheat germ agglutinin and concanavalin-A affected development. [ABSTRACT FROM AUTHOR]