Vitamin E, ?-tocopherol, diminishes ex vivo basophil response to dust mite allergen.
ABSTRACT: Epidemiologic studies suggest that dietary vitamin E is a candidate intervention for atopic disease. We used in vitro and ex vivo exposures to test the hypothesis that the most common dietary isoform of vitamin E, ?-tocopherol (?T), could suppress Fc?RI-mediated basophil activation. Rat basophilic leukemia (RBL)-SX38 cells that express human Fc?RI were treated with or without ?T, followed by stimulation with ?-IgE. In the ex vivo study, 20 Der f 1-allergic volunteers consumed a ?T-enriched supplement for 7 days. Their basophils were challenged ex vivo with ?-IgE and graded doses of Der f 1 before and after the supplementation period. ?t treatment of RBL-SX38 cells significantly reduced basophil degranulation and de novo TH2 cytokine production. Daily consumption of a ?T-rich supplement by dust mite-allergic volunteers reduced basophil activation after ex vivo dust mite challenge. Vitamin E supplements rich in ?T may be useful adjuncts in decreasing atopic disease.
Project description:Recently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST).bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human Fc?RI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting.Only 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions.Although bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.
Project description: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.
Project description: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.
Project description:Atopic dermatitis (AD) is a chronic recurrent inflammatory skin disease. There are a lot of evidences on the importance of vitamin D and house dust mite (HDM) allergens in the etiology and course of AD. The objectives of this study are to evaluate the relation between vitamin D3 level and house dust mites (HDM) Dermatophagoidspecies sensitization in pathogenesis of atopic dermatitis. Cross-sectional design study was conducted on 50 atopic dermatitis patients. Blood analysis were done to determine level of vitamin D3, total IgE by fluorescent immunoassay & specific IgE for HDM (d1, d2) & other inhalant allergens by ELISA test. There was significant high negative correlation with the specific IgE for HDM (r?=??-0. 62, p?<?0.001) and vitamin D3. & there was non-significant minimal negative correlation with the specific IgE and other inhalant allergens (r?=?-?0.10, p?>?0.05). There was a statistically significant relation between level of vitamin D3 and atopic dermatitis severity and sensitization to HDM and other allergens.
Project description:Basophils are involved in manifestations of hypersensitivity, however, the current understanding of their propensity for activation and their prognostic value in cancer patients remains unclear. As in healthy and atopic individuals, basophil populations were identified in blood from ovarian cancer patients (n = 53) with diverse tumor histologies and treatment histories. Ex vivo basophil activation was measured by CD63 expression using the basophil activation test (BAT). Irrespective of prior treatment, basophils could be activated by stimulation with IgE- (anti-Fc?RI and anti-IgE) and non-IgE (fMLP) mediated triggers. Basophil activation was detected by ex vivo exposure to paclitaxel, but not to other anti-cancer therapies, in agreement with a clinical history of systemic hypersensitivity reactions to paclitaxel. Protein and gene expression analyses support the presence of basophils (CCR3, CD123, Fc?RI) and activated basophils (CD63, CD203c, tryptase) in ovarian tumors. Greater numbers of circulating basophils, cells with greater capacity for ex vivo stimulation (n = 35), and gene signatures indicating the presence of activated basophils in tumors (n = 439) were each associated with improved survival in ovarian cancer. Circulating basophils in cancer patients respond to IgE- and non-IgE-mediated signals and could help identify hypersensitivity to therapeutic agents. Activated circulating and tumor-infiltrating basophils may be potential biomarkers in oncology.
Project description:Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1-3, 6, 7, 10, 11, 13-18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (?)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ?20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ?5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of ?-actinin as an allergen.
Project description:Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.
Project description:Allergens from house dust mites (HDM) are a common cause of asthma. Der p and Der f from Dermatophagoides sp. are strong immunogens in humans. Allergen extracts are used to study T helper (Th2) cell responses to HDM, which are implicated in the development and regulation of allergic disease.To define an epitope mixture that recapitulates, and might substitute for, HDM extract in terms of detecting and characterizing Th2 cell responses.Peripheral blood mononuclear cells (PBMC) from 52 HDM allergic and 10 non-allergic individuals were stimulated with HDM extracts and assayed with a set of 178 peptides spanning mite allergens group Der p 1, 2, 23 and Der f group 1 and 2 allergens. A pool of the most dominant T cell epitopes identified in the present study and from published literature was assembled and tested for ex vivo T cell responses. Correlation with HDM-specific IgE titres was examined.Patterns of T cell reactivity to Der p and Der f - derived peptides revealed a large number of epitopes. Clear patterns of immunodominance were apparent, with HDM allergen group 1 and 2 dominant over group 23. Furthermore, within a given antigen, 6-11 epitopes accounted for the vast majority of responses. Based on these results and published data, a comprehensive dust mite pool (DMP) of epitopes was designed and found to allow detection of ex vivo T cell responses. DMP ex vivo reactivity correlated with HDM-specific IgE titres and was similar to that detected with commonly used HDM extracts. Ex vivo DMP stimulation was associated with a predominant Th2 response in allergic donors, and minor reactivity of T cells producing IFNÎ³, IL17 and IL10.A detailed map of Der p and Der f antigens defined a pool of epitopes that can be used to detect ex vivo HDM responses.
Project description:Engineered nanomaterials (ENMs) interact with different biomolecules as soon as they are in contact, resulting in the formation of a biomolecule 'corona'. Hence, the 'corona' defines the biological identity of the ENMs and could affect the response of the immune system to ENM exposure. With up to 40 % of the world population suffering from type I allergy, a possible modulation of allergen effects by binding to ENMs is highly relevant with respect to work place and consumer safety. Therefore, the aim of this present study was to gain an insight into the interactions of gold nanoparticles with different seasonally and perennially occurring outdoor and indoor allergens.Gold nanoparticles (AuNPs) were conjugated with the major allergens of birch pollen (Bet v 1), timothy grass pollen (Phl p 5) and house dust mite (Der p 1). The AuNP-allergen conjugates were characterized by means of TEM negative staining, dynamic light scattering (DLS), z-potential measurements and hyperspectral imaging. Furthermore, 3D models were constructed, based on the characterization data, to visualize the interaction between the allergens and the AuNPs surface. Differences in the activation of human basophil cells derived from birch/grass pollen- and house dust mite-allergic patients in response to free allergen and AuNP-allergen conjugates were determined using the basophil activation assay (BAT). Potential allergen corona replacement during BAT was controlled for using Western blotting. The protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was assessed, by an enzymatic activity assay and a cellular assay pertaining to lung type II alveolar epithelial cell tight junction integrity.The formation of a stable corona was found for all three allergens used. Our data suggest, that depending on the allergen, different effects are observed after binding to ENMs, including enhanced allergic responses against Der p 1 and also, for some patients, against Bet v 1. Moreover elevated protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was found.In summary, this study presents that conjugation of allergens to ENMs can modulate the human allergic response, and that protease activity can be increased. Graphical Abstract Cross-linking of IgE receptors and degranulation of human basophils due to epitope alignment of nanoparticle-coated allergens.
Project description:Tropomyosins represent clinically relevant seafood allergens but the role of mite tropomyosin, Der p 10, in house dust mite (HDM) allergy has not been studied in detail.To express and purify a recombinant Der p 10 with equivalent IgE reactivity as natural Der p 10 and to evaluate its IgE reactivity and allergenic activity in HDM-allergic patients.rDer p 10 was expressed in Escherichia coli, purified and characterized by mass spectrometry and circular dichroism. It was tested for IgE reactivity in 1322 HDM-allergic patients. Detailed IgE-reactivity profiles to six HDM allergens (Der p 1, 2, 5, 7, 10, 21) were established for subgroups of Der p 10-positive and -negative patients. The allergenic activity of rDer p 10 was evaluated in basophil degranulation experiments.rDer p 10 is an ?-helical protein sharing IgE epitopes with nDer p 10. It is recognized by 15.2% of HDM-allergic patients. Der p 10-negative patients were primarily sensitized to Der p 1 and/or Der p 2, whereas Der p 10-positive patients reacted to several other HDM allergens besides the major allergens (Der p 1, Der p 2) or showed a rather selective Der p 10 reactivity. The allergenic activity of Der p 10 was generally low but patients could be identified who suffered from clinically relevant HDM allergy due to Der p 10 sensitization.Der p 10 may be a diagnostic marker for HDM-allergic patients with additional sensitization to allergens other than Der p 1 and Der p 2. Such patients may require attention when allergen-specific immunotherapy is considered.