Novel Approaches for Inhibiting the Indoor Allergen Der f 2 Excreted from House Dust Mites by Todomatsu Oil Produced from Woodland Residues.

House dust mite (HDM) is a globally ubiquitous domestic cause of allergic diseases. There is a pressing demand to discover efficient, harmless, and eco-friendly natural extracts to inhibit HDM allergens that are more likely to trigger allergies and challenging to be prevented entirely. This study, therefore, is aimed at assessing the inhibition of the allergenicity of major HDM allergen Der f 2 by todomatsu oil extracted from residues of Abies Sachalinensis. The inhibition was investigated experimentally (using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) and in silico using molecular docking. The results showed that todomatsu oil inhibits the allergenicity of Der f 2 by reducing its amount instead of the IgG binding capacity of a single protein. Moreover, the compounds in todomatsu oil bind to Der f 2 via alkyl hydrophobic interactions. Notably, most compounds interact with the hydrophobic amino acids of Der f 2, and seven substances interact with CYS27. Contrarily, the principal compounds fail to attach to the amino acids forming the IgG epitope in Der f 2. Interestingly, chemical components with the lowest relative percentages in todomatsu oil show high-affinity values on Der f 2, especially ß-maaliene (-8.0 kcal/mol). In conclusion, todomatsu oil has been proven in vitro as a potential effective public health strategy to inhibit the allergenicity of Der f 2.

Dietary shifts have consequences for the repertoire of allergens produced by the European house dust mite.

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.

Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

A novel nanostructured biosensor for the detection of the dust mite antigen Der p2.

The group 2 allergen, Der p2, has been reported to activate innate toll-like receptors (TLRs) on respiratory epithelial cells and thus aggravate respiratory diseases. In this study, a highly sensitive nanostructured biosensor based on a 3D sensing element with uniformly deposited gold nanoparticles is proposed for the detection of the dust mite antigen Der p2. The barrier layer comprises an anodic aluminum oxide (AAO) film which is used as the template in this highly sensitive nanostructured biosensor. Simple electrochemical deposition without reducing agent and stabilizer was enough to uniformly synthesize gold nanoparticles on the surface of the barrier layer. The size and the distribution density of the nanoparticles can be well controlled by the applied potential during electrochemical deposition. Following this procedure, the dust mite monoclonal antibodies (IgG) were then immobilized through the 11-MUA (11-mercaptoundecanoic acid), (1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide)/(N-hydroxysuccinimide) self-assembled monolayer approach for the dust mite antigen Der p2 detection. The detection limit of the proposed 3D gold nanoparticle-based nanostructured biosensor was examined using electrochemical impedance spectroscopy analysis and found to be 1 pg/mL. The dynamic range was found to be 5 µg/mL. The proposed nanostructured biosensor would be useful for fast detection of rare molecules in a solution.

Oxidized cellulose binding to allergens with a carbohydrate-binding module attenuates allergic reactions.

Grass and mite allergens are of the main causes of allergy and asthma. A carbohydrate-binding module (CBM) represents a common motif to groups I (ß-expansin) and II/III (expansin-like) grass allergens and is suggested to mediate allergen-IgE binding. House dust mite group II allergen (Der p 2 and Der f 2) structures bear strong similarity to expansin's CBM, suggesting their ability to bind carbohydrates. Thus, this study proposes the design of a carbohydrate-based treatment in which allergen binding to carbohydrate particles will promote allergen airway clearance and prevent allergic reactions. The aim of the study was to identify a polysaccharide with high allergen-binding capacities and to explore its ability to prevent allergy. Oxidized cellulose (OC) demonstrated allergen-binding capacities toward grass and mite allergens that surpassed those of any other polysaccharide examined in this study. Furthermore, inhalant preparations of OC microparticles attenuated allergic lung inflammation in rye grass-sensitized Brown Norway rats and OVA-sensitized BALB/c mice. Fluorescently labeled OC efficiently cleared from the mouse airways and body organs. Moreover, long-term administration of OC inhalant to Wistar rats did not result in toxicity. In conclusion, many allergens, such as grass and dust mite, contain a common CBM motif. OC demonstrates a strong and relatively specific allergen-binding capacity to CBM-containing allergens. OC's ability to attenuate allergic inflammation, together with its documented safety record, forms a firm basis for its application as an alternative treatment for prevention and relief of allergy and asthma.

Protein chips for detection of mite allergens using Kunitz-type protease inhibitors.

Stored-food and house-dust arthropods include many species of mites and beetles that affect human health. For diagnostic tests proteases such as trypsin are utilized as they are indicators of the presence of allergen contaminants in food. We recently characterized Kunitz-type protease inhibitors (KPIs) from Solanum palustre. Here we studied biotechnological applications of KPI-B1 and -B4. We manufactured a protein chip with immobilized KPI-B1 and -B4 and showed trypsin/chymotrypsin-binding specificity, indicating that the recombinant proteins have protease selectivity. We employed the protein chip to capture mite proteins belonging to the protease family with polyclonal anti-mite antibodies. The mite diagnostic chip can be useful for detecting mite allergens.

[Flow cytometric basophils activation test as a method of allergy diagnosis]. / Cytometryczna ocena aktywacji bazofilów w diagnostyce chorób alergicznych.

For several years the incidences of allergic diseases and anaphylactic reactions have been increasing dramatically. Classical method of allergy diagnosis - skin prick test in some situations can provoke life-threatening reactions. Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis in those patients. CD 63 and CD203c have recently been demonstrated as a specific activation markers of basophils that are rapidly up-regulated after allergen challenge in sensitized patients. Although flow-cytometry methods are quite sophisticated and expensive, it could be a good alternative in patients at risk of severe anaphylactic reactions or with contradictory test results.

Oral administration of a hop water extract ameliorates the development of dermatitis induced by the periodical topical application of a mite antigen in atopic dermatitis model NC/Nga mice.

We investigated the inhibitory effect of an oral administration of a hop water extract (HWE) on the development of dermatitis by using NC/Nga atopic dermatitis model mice. The induction of allergic dermatitis was conducted by tape-stripping and topical application of a mite antigen (Dermatophagoides farinae) on to the ear once a week for 10 weeks. HWE was orally administered at a dose of 100 or 500 mg/kg. The total immunoglobulin E (IgE) concentration in serum and the ear thickness were periodically examined. Finally, the antigen-specific IgE level in the serum and the production of interleukin (IL)-4, IL-12 and interferon (IFN)-gamma from splenocytes and cervical lymph node cells were measured. The oral administration of HWE significantly inhibited the increase of total IgE production and ear swelling throughout the experimental period. The production of IL-12 was significantly lower in the HWE administered group than in the control group. The results suggest that the intake of HWE may be effective in preventing and alleviating the development of atopic dermatitis-like skin disease.

The squamous cell carcinoma antigen 2 inhibits the cysteine proteinase activity of a major mite allergen, Der p 1.

The squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 belong to the ovalbumin-serpin family. Although SCCA1 and SCCA2 are closely homologous, these two molecules have distinct properties; SCCA1 inhibits cysteine proteinases such as cathepsin K, L, and S, whereas SCCA2 inhibits serine proteinases such as cathepsin G and human mast cell chymase. Although several intrinsic target proteinases for SCCA1 and SCCA2 have been found, the biological roles of SCCA1 and SCCA2 remain unknown. A mite allergen, Der p 1, is one of the most immunodominant allergens and also acts as a cysteine proteinase probably involved in the pathogenesis of allergic diseases. We have recently shown that both SCCA1 and SCCA2 are induced by two related Th2-type cytokines, IL-4 and IL-13, in bronchial epithelial cells and that SCCA expression is augmented in bronchial asthma patients. In this study, we explored the possibility that SCCA proteins target Der p 1, and it turned out that SCCA2, but not SCCA1, inhibited the catalytic activities of Der p 1. We furthermore analyzed the inhibitory mechanism of SCCA2 on Der p 1. SCCA2 contributed the suicide substrate-like mechanism without formation of a covalent complex, causing irreversible impairment of the catalytic activity of Der p 1, as SCCA1 does on papain. In addition, resistance to cleavage by Der p 1 also contributed to the inhibitory mechanism of SCCA2. These results suggest that SCCA2 acts as a cross-class serpin targeting an extrinsic cysteine proteinase derived from house dust mites and that it may have a protective role against biological reactions caused by mites.