Cloning, sequence analysis and expression in E. coli of the group 3 allergen of Dermatophagoides farinae

Background The dust mites, which are mostly represented by Dermatophagoides spp. （Acari： Pyroglyphidae）, are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites. Methods Total RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a （＋）, expressed in E. coil BL21 at the aid of the inducer isopropyI-D-thiogalactopyranoside （IPTG）. The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software. Results The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f3 cDNA clones in comparison with the reference （GenBank Accession No. AY283291）, which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites （53-68 AA, 108-122 AA and 205-217 AA）, one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites. Conclusions Der f3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f3 gene but this needs to be further confirmed in the future.

House dust mite allergen Der f enhanced bronchial epithelial cell cytokine expression

The house dust mites (Dermatophagoides farinae, Derf) are the major source of aeroaller gens implicated in the expression of atopic disorders, including asthma, allergic rhinitis and atopic dermatitis. In particular, strong circumstantial evidence suggests that house dust mite antigens are important precipitating factors of asthma. Many house dust mite allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines from bronchial epithelial cells. To investigate whether Der f allergen proteases induced cytokine production from the epithelial cell line BEAS-2B, BEAS-2B cells were cultured with 4 different concentrations of Derf (0.02, 0.2, 2, 20μg/ml) for 24-96 h, after which supernatants were assayed for interleukin (IL)-6 and IL-8 with ELISA. Reverse transcription-PCR was also performed. The cell sheets were intact throughout the observation in control group without any exposure to Der f antigen. In the experimental groups cells treated with Der f allergen showed changes in the anchorage status of the monolayer. There was a significant increase in the level of cytokine production compared with the untreated sample. The release of IL-6 and IL-8 increased in a concentration-dependent manner (P <0.05, respectively) with the addition of increasing dosage of Der f to the cell sheets. Levels of IL-6 and IL-8 began to rise at 24 h and 48 h after allergen exposure, and they increased significantly in the supematants at 72 h and 96 h. At the same time the concentration dependence of induction of IL-6 and IL-8 expression as well as an increase in the expression of IL-6 and IL-8 mRNA manifested evidently. HDM-induced airway inflammation may include Der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. It is suggested that IL-6 and IL-8 production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma.

Topical Application of Cudrania tricuspidata Stem Extract Inhibits Atopic Dermatitis-Like Skin Lesions in an NC/Nga Mouse Model: An Experimental Animal Study

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by elevated immunoglobulin E (IgE), mast cell infiltration and skin lesions including pruritus, erythema and eczema. Cudrania tricuspidata extracts have been clinically administered for a long time in the East Asia including Korean and China as a home-remedy to diminish the inflammation of gastritis and hepatitis. To examine whether it works on AD or not, an AD-like animal model was experimented in this study. AD was induced by applying Dermatophagoides farinae (D. farinae) extract to the backs of 9-week old NC/Nga mice for 21 days. Following this, an ethanol extract of C. tricuspidata stems (EECT) was applied topically for 14 days to the sensitized skin, while distilled water was used as a control (EECT0 mice). Anti-AD effects of EECT were evaluated using scores for AD-like skin lesions, serum IgE levels and mast cell counts in the skin dermal layers to assess inflammation. Topically applied ethanol extract of Cudrania tricuspidata stems (EECT 7.5, 25 and 75 mg/mL) markedly reduced AD-like skin lesions after 4 days (by 30.1%, 31.4% and 38.5%, respectively) and also after 14 days (by 63.6%, 66.1% and 49.6%, respectively), while distilled water improved AD by 17.8% and 38.7%, respectively (p D. farinae extract (p = 0.003) and EECT attenuated the mast cell overproduction, and reduced mast cell degranulation markedly. Attenuation was most obvious in the early stage of EECT treatment when the AD was most acute.

Allergen micro-array detection of specific IgE-reactivity in Chinese allergy patients

Background Allergen micro-arrays are powerful tools for screening of serum IgE-reactivity. In this study allergen micro-arrays were used to identify dominating IgE-binding allergens and cross-reactivity patterns among selected Chinese allergy patients. Methods The study was conducted using patient sera from the cities of Guangzhou, Nanjing, Chengdu and Shenyang. In total 100 sera with Dermatophagoides pteronyssinus （Der p） specific IgE-levels higher than 50 kU/L were selected for testing against 103 individual allergens. Results Among 100 selected patients, 95% showed IgE-reactivity towards house-dust mite allergens Dermatophagoides farinae （Der f） 1, Der f 2 and Der p 2 and 94% were IgE positive against Der p 1, and 60% of sera contained IgE reacting against allergen Euroglyphus maynei （Eur m） 2. IgE against cat allergen, Felisdomesticus （Fel d） 1, was seen in 20%. Only 2% showed specific IgE-reactivity to Der p 10, a panallergen belonging to the tropomyosin family. Serum IgE-reactivity towards other allergens was in general low. IgE-reactivity against pollen allergens showed geographic differences. Conclusions This study clearly confirms that group 1 and group 2 are major allergens of house dust mites. These selected house-dust mite allergy patients are close to being mono-sensitized. Der p 10 is not an important allergen for cross-reactivity. Specific IgE-sensitization towards pollen allergens is low in southern China compared to other regions. The prevalence of food and stinging insect allergens known to give rise to IgE-mediated cross-reactivity is 2% or less.