Acaricidal activities of some essential oils and their monoterpenoidal constituents against house dust mite,Dermatophagoides pteronyssinus (Acari:Pyroglyphidae)

The acaricidal activities of fourteen essential oils and fourteen of their major monoterpenoids were tested against house dust mites Dermatophagoides pteronyssinus. Five concentrations were used over two different time intervals 24 and 48 h under laboratory conditions. In general, it was noticed that the acaricidal effect based on LC50 of either essential oils or monoterpenoids against the mite was time dependant. The LCso values were decreased by increasing of exposure time. Clove, matrecary, chenopodium, rosemary, eucalyptus and caraway oils were shown to have high activity. As for the monoterpenoids, cinnamaldehyde and chlorothymol were found to be the most effective followed by citronellol. This study suggests the use of the essential oils and their major constituents as ecofriendly biodegradable agents for the control of house dust mite, D. pteronyssinus.

Comparison of Three Methods of Protein Extraction from Dermatophagoides Pteronyssinus for Two-dimensional Electrophoresis

Obiective To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis （2-DE） analysis. Methods The extracts of Dermatophagoides pteronyssinus were prepared with Coca＇s solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid （BCA） assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis. Results The concentrations of extracted protein by methods of Coca＇s solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight （LMW） range could be detected with the Coca＇s solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight （MMW） protein spots were absent. Several MMW protein spots （174-178 kD and 133 kD） and more LMW protein spots were detected withTrizol reagent method. Conclusions Among Coca＇s solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.

Comparison and Evaluation of Several Dermatophagoides Pteronyssinus Allergen Extracts for Skin Prick Test

Objective To evaluate the significance of several Dermatophagoides pteronyssinus allergen extracts for skin prick test(SPT) in patients allergic to Dermatophagoides pteronyssinus.Methods Two hundred and nineteen patients enrolled in Peking Union Medical College Hospital underwent SPT and serum specific IgE assay to detect the Dermatophagoides pteronyssinus allergen.Three kinds of house dust mite allergen extracts were used for SPT,including the Dermatophagoides pteronyssinus extract prepared by our laboratory(group A),standardized Dermatophagoides pteronyssinus extract(group B),and mixed extracts of Dermatophagoides pteronyssinus and Dermatophagoides farinae(group C).Human serum specific IgE result was regarded as the reference standard for diagnosis of Dermatophagoides pteronyssinus allergy.The receiver operating characteristic(ROC) curve was used to evaluate the diagnostic performance of SPT with the extracts of three groups.Results SPT results showed that the median wheal diameter of group A,group B,and group C was 0.43,0.35,and 0.28 cm,respectively,with significant difference among three groups(P<0.05).The difference was significant between group A and B(P<0.01) as well as group A and C(P<0.01),but not between group B and C(P>0.05).There was no local urticaria or systemic allergic reactions following the procedure of SPT.Local reaction was observed in 5 patients and delayed reaction was in 2 patients of group A.As for group B and C,local reaction occurred in 3 cases and delayed reaction in 2 cases in each group.The area under ROC curve of SPT with extract in group A,group B,and group C was 0.765,0.801,and 0.782,respectively.Based on the detection results of serum specific IgE,the sensitivity of SPT in diagnosis of Dermato-phagoides pteronyssinus allergy with extract of group A,group B,and group C was 92.4%,87.0%,and 81.5%,and the specificity was 60.6%,73.2%,and 74.8%,respectively.Conclusion The Dermatophagoides pteronyssinus extract for SPT prepared by our laboratory offers good sensitivity and specificity comparable to commercially available allergen extracts,and it may be an appropriate candidate for clinical screening and diagnosis of Dermatophagoides pteronyssinus allergy.

Diagnostic accuracy and safety of Dermatophagoides pteronyssinus extracts used for skin prick test

Background:Dermatophagoides pteronyssinus is a common allergen causing allergic diseases in China.The aim of this study was to evaluate the efficacy and safety of D.pteronyssinus extracts produced by Peking Union Medical College Hospital(PUMCH)for the skin prick test(SPT)in the diagnosis of D.pteronyssinus allergy.Methods:A total of 910 subjects with allergic diseases were prescribed D.pteronyssinus SPT and specific sIgE(sIgE)test among the Outpatients of Department of Allergy,PUMCH from August 10,2015 to August 30,2017.Receiver operating characteristic curve(ROC)analysis was performed according to the results of D.pteronyssinus-sIgE detection.The accuracy of D.pteronyssinus extracts used for SPT in the diagnosis of D.pteronyssinus allergy was evaluated under different cutoff values.Adverse events after SPT were recorded to evaluate safety.Results:There were 796 and 618 subjects in the full analysis set(FAS)and the per protocol set(PPS),respectively.The areas under the curve of FAS and PPS were 0.871 and 0.873,respectively.According to the ROC of PPS,the optimal and 95%specificity diagnostic cutoff values of D.pteronyssinus SPT mean wheal diameter were 3.25 and 3.75 mm,respectively.No adverse events occurred.Conclusion:The extracts of D.pteronyssinus for SPT were simple,highly accurate,and safe and should be considered for recommendation in the clinical diagnosis of D.pteronyssinus allergy.

Indoor Allergen Levels and Household Distributions in Nine Cities Across China

Objective Chinese allergic subjects have high levels of sensitization to house dust mite （HDM） and other indoor allergens. This study quantifies common indoor allergen levels in Chinese households. Methods Dust samples were collected from nine cities. Major allergens Der p 1 and Der f I from Dermatophagoides pteronyssinus and D. farinae, and specific antigens of Blomia tropicalis, Tyrophagus putrescentiae, Acarus siro, and cockroach species Blattella germanica and Periplaneta americana were measured by ELISA.Results HDM allergens were found in dust samples from bedding in 95% of the Chinese households. The median levels varied from 〈0.006 to 9.2 μg/g of dust, depending on the city. The percentages of households having HDM allergen levels associated with the risk of developing allergy sensitization and asthma were 65% and 25%, respectively. Specific antigens of the storage mite and cockroach were only found in samples from the southern and tropical regions of China. Levels of mite allergens were generally higher in samples from bedding compared to samples from the living room, even for storage mites, whereas levels of cockroach antigens were higher in the living room samples.Conclusion HDM allergens are present in bedding dust samples from most Chinese households. Cities in southern and central China have relatively high levels of HDM major allergens compared to cities in northern and western China. Antigens of storage mites and cockroaches are not as common as HDM allergens.

Resistance of Commercial Bed Covers against Faecal Pellets of House Dust Mites:Behaviour of Seams and Zippers

This study evaluated the allergen impermeability against airborne allergens of dust mite droppings through all parts of commercial bed covers, including surface seams and zippers. Specimens were taken from places with and without seams and zipper. A novel penetration cell was developed to expose the specimens to an inoculum of purified mite droppings that was assessed for its allergen content Der p1 prior to the penetration tests. Using covers of different construction and material, the penetration level increased significantly in the presence of seams and zippers and could reach up to 6% depending on the seam’s/zipper’s characteristics and quality. Therefore, zippers and seams have to be considered as access points for the penetration of mite droppings. As for the penetration of airborne mite particles through the zipper, the penetration level was greatly attenuated by the presence of a cover strip. Depending on the respective quality and the construction type, the mite allergen Der p1 penetrated most likely through the zipper and seams of the specimens, already after a single laundry cycle. Hence, laundry may compromise the barrier performance and proves to be an important quality feature. In all samples, the textile surface showed sufficient allergen impermeability. Our conclusions provide recommendations to both manufacturers and users.

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.

DNA vaccine encoding Der p 2 allergen generates immunologic protection in recombinant Der p 2 allergen-induced allergic airway inflammation mice model

Background DNA immunization is a promising novel type of immunotherapy against allergy. An estimated 79.2% patients with asthma, wheezing and/or rhinitis suffer from Dermatophagoides pteronyssinus group 2 (Der p 2) allegen. The aim of the present study was to determine whether DNA vaccine encoding Der p 2 could generate immunologic protection in recombinant Der p 2 (rDer p 2) allergen-induced allergic airway inflammation mice model and to understand the role of DNA vaccination in specific-allergen immunotherapy for asthma. Methods After DNA vaccination, BALB/c mice were sensitized by intraperitoneal injection (i.p) and challenged by intranasal instillation of rDer p 2. The lung tissues were assessed using hematoxylin and eosin. Mucus-producing goblet cells were identifed using periodic acid-Schiff(PAS)/alcian blue. The total cell number and composition of bronchoalveolar lavage samples were determined. The levels of the cytokines IL-4 and IFN-γ, as well as IgE and IgG_(2a)in the serum were determined by enzyme-linked immunosorbent assay. Allergen-specific IL-4 and IFN-γ production by spleen cells were also measured by enzyme-linked immunosorbent assay. Expression of signal transducer and activator of transcription 6 (STAT6) in splenocytes were determined by Western blot. Results DNA vaccine encoding Der p 2 allergen inhibited extensive infiltration of inflammatory cells and production of mucin induced by allergen. The influx of eosinophils into the lung interstitium was significantly reduced after administration of DNA vaccine. Significant reductions of IL-4 and increase in levels of IFN-γ in bronchoalveolar lavage fluid were observed. The allergen-specific IgE was markedly decreased in mice receiving DNA vaccination. Allergen could induce higher IFN-γ, weaker IL-4 in cultured spleen cells from mice receiving DNA vaccine. DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen. Conclusion These results indicated that DNA vaccine encoding Der p 2 allergen generates immunologic protection in recombinant Der p 2 allergen-induced allergic airway inflammation mice model with regulating the immune response towards a Th1-type reaction.

Induction of Th1-Type Immune Response by Chitosan Nanoparticles Containing Plasmid DNA Encoding House Dust Mite Allergen Der p2 for Oral Vaccination in Mice

This study was to prepare the chitosan-pDer p 2 nanoparticles and to investigate the effect of chitosan-DNA nanoparticles on immune response in mice by oral delivery of chitosan-DNA nanoparticles. The nanoparticles were synthesized by complexing chitosan with plasmid DNA. The DNA was fully complexed into chitosan-DNA nanoparticles, suggesting a 100% encapsulation efficiency. Chitosan-DNA complex renders a significant protection of the plasmid. No effect on cell viability was observed in both cell types and average cell viability over 100% was obtained. Oral gene delivery with chitosan-DNA nanoparticles can generate a higher level expression of gene in vivo. Oral chitosan-pDer p 2 nanoparticles in BALB/c mice can induce IFN-γ in serum and prevent subsequent sensitization of Th2 cell-regulated specific IgE responses. The data indicate that the oral administration of chitosan-pDer p 2 nanoparticles results in the expression of Der p 2 in the epithelial cells of both stomach and small intestine and the induction of Th1-type immune response. Cellular ＆ Molecular Immunology.

Therapeutic Effects of DNA Vaccine on Allergen-Induced Allergic Airway Inflammation in Mouse Model

Vaccination with DNA encoding Dermatophagoides pteronyssinus group 2 （Der p 2） allergen previously showed its effects of immunologic protection on Der p 2 allergen-induced allergic airway inflammation in mice. In present study, we investigated whether DNA vaccine encoding Der p 2 could exert therapeutic role on allergen-induced allergic airway inflammation in mouse model and explored the mechanism of DNA vaccination in asthma specific-allergen immunotherapy. After sensitized and challenged by Der p 2, the BALB/c mice were immunized with DNA vaccine. The degrees of cellular infiltration were scored. IgE levels in serum and IL-4/IL-13 levels in BALF were determined by ELISA. The lung tissues were assessed by histological examinations. Expressions of STAT6 and NF-kB in lung were determined by immunohistochemistry staining. Vaccination of mice with DNA vaccine inhibited the development of airway inflammation and the production of mucin induced by allergen, and reduced the level of Der p 2-specific IgE level. Significant reductions of eosinophil infiltration and levels of IL-4 and IL-13 in BALF were observed after vaccination. Further more, DNA vaccination inhibited STAT6 and NF-kB expression in lung tissue in Der p 2-immunized mice. These results indicated that DNA vaccine encoding Der p 2 allergen could be used for therapy of allergen-induced allergic airway inflammation in our mouse model. Cellular ＆ Molecular Immunology.

Dust mite allergen-specific immunotherapy increases IL4 DNA methylation and induces Der p-specific T cell tolerance in children with allergic asthma

Allergen-specific immunotherapy(allergen-SIT)is a highly effective treatment for children with allergic asthma(AA),an immune-mediated chronic disease leading to bronchial muscle hypertrophy and airway obstruction in response to specific allergens.T helper cells and secreted cytokines play important roles in the pathogenesis of asthma,and epigenetic modulation controls genes important for T cell development and cytokine expression.This study evaluated T helper cell-secreted cytokines and DNA methylation patterns in children treated with Dermatophagoides pteronyssinus(Der p)allergen-SIT.Our results showed that after Der p challenge,peripheral blood mononuclear cells(PBMCs)from the SIT group,compared with the non-SIT AA group,produced lower levels of IL-4,IL-5 and IL-2.The SIT group,compared with the AA group,exhibited decreased sensitivity to the Der p allergen,concurrent with IL-4 down-modulation due to increased promoter DNA methylation,as estimated in PBMCs.Our results showed that SIT decreased IL-4 and IL-5,and inhibited T cell proliferation,by inhibiting IL-2 production after the specific allergen challenge.These results suggest that decreased IL-2 production and increased IL-4 cytokine promoter methylation is a potential mechanism of Der p-specific allergen desensitization immunotherapy.

DNA vaccine encoding Der p2 allergen down-regulates STAT6 expression in mouse model of allergen-induced allergic airway inflammation

Background Activation of signal transducer and activator of transcription 6 （STAT6） plays a critical role in the late phase of Th2-dependent allergy induction. STAT6 is essential to Th2 cell differentiation, recruitment, and effector function. Our previous study confirmed that DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen. In the present study, we determined whether DNA vaccine encoding Dermatophagoides pteronyssinus group 2 （Der p2 ） could down-regulate the expression and activation of STAT6 in lung tissue from asthmatic mice. Methods After DNA vaccine immunization, BALB/c mice were sensitized by intmperitoneal injection and challenged by intranasal instillation of rDer p2. The levels of the cytokines IL-4 and IL-13 in BAL fluid were measured by enzyme-linked immunosorbent assay. The lung tissue was assessed by immunohistochemical staining with anti-STAT6. The protein expression of STAT6 was determined by Western blot. The activation of STAT6 binding ability was analyzed with electrophoretic mobility shift assay. Results DNA vaccine encoding Der p2 allergen effectively decreased the levels of IL-4 and IL-13 in the asthmatic mice. Histological evidence and Western blot showed that the expression of STAT6 in the DNA treated mice was markedly attenuated. STAT6 binding to specific DNA motif in lung tissue from the gene vaccinated mice was inhibited. Condusion DNA vaccine encoding Der p2 prevents allergic pulmonary inflammation probably by inhibiting the STAT6 signaling pathway in mice with Der p2 allergen-induced allergic airway infammafion.