BACKGROUND: The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated. OBJECTIVE: To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level. METHODS: Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments. RESULTS: Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients. CONCLUSION: Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.
- MeSH
- antigeny roztočů domácího prachu chemie genetika imunologie MeSH
- antisérum chemie MeSH
- bazofily cytologie účinky léků imunologie MeSH
- chitin chemie imunologie MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- interakční proteinové domény a motivy MeSH
- klonování DNA MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- králíci MeSH
- lidé MeSH
- proteiny členovců chemie genetika imunologie MeSH
- protilátky krev chemie izolace a purifikace MeSH
- Pyroglyphidae chemie ultrastruktura MeSH
- rekombinantní proteiny chemie genetika imunologie MeSH
- respirační alergie chemicky indukované imunologie patofyziologie MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented.
House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.
- MeSH
- antigeny roztočů domácího prachu chemie genetika imunologie MeSH
- atopická dermatitida epidemiologie imunologie MeSH
- biologické markery MeSH
- cirkulární dichroismus MeSH
- Dermatophagoides pteronyssinus genetika imunologie MeSH
- dítě MeSH
- dospělí MeSH
- imunoglobulin E krev imunologie MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- molekulární sekvence - údaje MeSH
- proteiny členovců MeSH
- protilátky krev imunologie MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- séroepidemiologické studie MeSH
- tropomyosin chemie genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
