尘螨过敏原的交叉反应性

尘螨是最主要的室内过敏原之一,随着社会生活的现代化,人们对室内居住环境的要求越来越高,由于环境因素而引起的一些过敏性疾病越来越受到人们的关注。本文主要综述了近几年来尘螨过敏原与其他多种螨类、软体动物（蜗牛）和甲壳类动物（虾）以及昆虫（蟑螂、衣鱼、摇蚊、石蚕蛾）等过敏原之间的交叉反应性,对临床过敏性疾病的诊断具有参考价值。     

Crystal structure and some properties of a major house dust mite allergen, Derf 2

Pyroglyphid house dust mites are a major source of allergens in house dust. Mite allergens sensitize and induce asthma, rhinitis, and eczema in a large portion of patients with allergic diseases. Here, the crystal structure of a major mite allergen, Derf 2, derived from Dermatophagoides farinae was solved by single isomorphous replacement method with anomalous scattering (SIRAS) at 2.1A resolution. The present study also demonstrated that the conformation of the allergen was critical in the determination of Th1/Th2 shift based on physicochemical and immunological analyses. This indicates that rigidly folded and singly dispersed structure is essentially required for the generation of Th2 type cells by the allergen, while conformational variant protein leads to Th1 skewing, irrespective of the same amino acid sequence. This structure/function relationship may allow us to develop a novel strategy for hyposensitization therapy in patients with allergic diseases triggered by house dust mite allergens.     

Optimisation of a screening procedure for house dust mite numbers in carpets and preliminary application to buildings

The relative performances of the mobility and heat escape methods for measuring house dust mite numbers in carpet has been evaluated. The optimum method was found to be the mobility method for 24 hours at ambient temperatures which exhibited a mite collection efficiency of approximately 30%. Measurements in three dwellings showed that the method should be applied to carpet at several locations in the living room and/or bedrooms as a general sampling procedure, or adjacent to loungeroom seating to determine the worst case scenario. Carpets using different types of fibre within the same dwelling should be assessed separately. For the limited number of dwellings investigated, wool carpets were found to exhibit higher mite numbers than nylon carpets, even when the former had been insect-resist treated. No house dust mites were found in the wool carpets of an office building which was mechanically ventilated and heated and achieved low indoor humidities in winter.     

粉尘螨变应原第7组分全长基因序列测定与分析

目的：获得粉尘螨变应原第7组分编码基因并了解其分子特征。方法：根据GenBank已公布的Derf7核酸序列设计引物,用RT—PCR扩增获得其编码基因,插入pMD19-T载体进行序列测定和生物信息学分析。结果：获得Derf7全长基因约642bp,与参考序列（GenBankAY283292）同源性达99．7％％,仅在249位”A→G”和439位“C→T”发生点突变,含1个完整的开放读码框。推测编码蛋白由213个氨基酸组成,信号肽序列位于1-17aa,亲水性指数为0．031,跨膜区域位于171—190aa,二级结构由一螺旋（57．28％）、延伸主链（6．57％）和无规卷曲（36．15％）组成;亚细胞定位于细胞质,含有N-糖基化位点1个（151-154aa）,蛋白激酶c磷酸化位点1个（193-195aa）,酪蛋白激酶Ⅱ磷酸化位点2个（155—158aaand173．176aa）。N端酰基化位点1个（97—102aa）。粉尘螨和屋尘螨变应原第7组分氨基酸序列相似度为86％,二者在螨类第7组分氨基酸序列构建出的分子进化树中聚成一簇。结论：获得了Derf7全长基因,为进一步获得其基因工程制品用于临床和实验研究奠定了基础。     

Short communication. Clustering reduces water loss by adult American house dust mites Dermatophagoides farinae (Acari: Pyroglyphidae)

We investigated male clustering behaviour in large bulk cultures of the American house dust mite, Dermatophagoides farinae. Aggregation in the colony was restricted to males only. The water loss rates for individual males in isolation and as a group of six were determined. Males in a group of six retained water nearly twice as effectively (they had lower rates of net water loss) than individuals. Clustering is thus an adaptive behaviour to help reduce water loss. Because the clustering response was observed exclusively by males, this behaviour may serve an additional purpose that involves a pheromone system.     

Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model

Background

Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.

Aim

We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice.

Methods

BALB/c mice were intranasally sensitized with 1 μg HDM on day 0 followed by daily intranasal challenge with PBS or 10 μg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 μg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected.

Results

Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue.

Conclusion

Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0171-0) contains supplementary material, which is available to authorized users.     

TSLP receptor is not essential for house dust mite-induced allergic rhinitis in mice

TSLP induces Th2 cytokine production by Th2 cells and various other types of cells, thereby contributing to Th2-type immune responses and development of allergic disorders. We found that house dust mite (HDM) extract induced TSLP production by nasal epithelial cells, suggesting that TSLP may be involved in development of HDM-induced allergic rhinitis (AR). To investigate that possibility in greater detail, wild-type and TSLP receptor-deficient (TSLPR^?/?) mice on the C57BL/6J background were repeatedly treated intranasally with HDM extract. The frequency of sneezing, numbers of eosinophils and goblet cells, thickness of submucosal layers, serum levels of total IgE and HDM-specific IgG1, and levels of IL-4, IL-5 and IL-13 in the culture supernatants of HDM-stimulated LN cells were comparable in the two mouse strains. Those findings indicate that, in mice, TSLPR is not crucial for development of HDM-induced AR.     

Molecular identification of house dust mites and storage mites

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR–RFLP of ITS2 region. With the established PCR–RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR–RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.     

Effectiveness of benzyl benzoate in elimination of house dust mites

The effect of elimination treatment with benzyl benzoate was examined in 30 adults with asthma caused by sensitivity to house dust mites. The patients were randomised into a control and an active group, who treated their mattresses with benzyl benzoate (Acarosan^®). The study lasted 12 months and the effect of the treatment was monitored by monthly dust sampling, analyzed for major allergens fromDermatophagoides pteronyssinus andfarinae by the ELISA method and for guanine by the Acarex^® method. The clinical effect was assessed by measuring lung function, daily peak flows, symptoms and medicine consumption as well as skin prick tests, and specific IgE to mite allergens. For both groups, a significant decrease was observed in house dust mite allergens, but there was no significant difference between the groups. No considerable differences were observed in clinical parameters within or between the groups. A good correlation was observed between ELISA and Acarex^®. but the latter showed a major variation in the different classes. In conclusion. treatment of mattresses with benzyl benzoate had no effect in a group of patients with asthma due to house dust mite allergy. Regular vacuum cleaning of the bed may reduce house dust mite allergen exposure.     

尘螨连续石蜡切片的制备及染色技术

研究尘螨连续石蜡切片的制备技术。利用琼脂预包埋后再以塑化石蜡包埋,经切片和HE染色,获得结构完整、定位准确、染色清晰的连续切片。探讨制片过程中一些步骤的改进和注意事项,为尘螨显微结构的形态学研究提供可能,也为免疫组化、原位PCR及过敏性疾病尘螨特异性变应原的定位等研究奠定良好的基础。     

Seasonal prevalence of allergenic mites in house dust of Kolkata Metropolis,India

During the past few decades, house dust mites have attracted worldwide interest among medical entomologists and acarologists because of their importance in causing nasobronchial allergic disorders in human beings. House dust mites are present throughout the year; however, their relative densities differ in different seasons and habitats. Because the prevalence of house dust mite allergen is important epidemiologically and clinically, detailed knowledge on the seasonal abundance of important allergenic mites is of great importance for better understanding of the pathogenesis of the disease. In view of this, a systematic survey was carried out on the prevalence of total mites and four common allergenic mites in the city of Kolkata for two consecutive years. Both bed and bedroom floor dust were collected separately from homes inhabited by asthmatic patients situated in different corners of the city on monthly basis from January 2004 to December 2005. The population levels of total mites and four common allergenic mites, namely Dermatophagoides pteronyssinus, D. farinae, Austroglycyphagus geniculatus, and Blomia tropicalis separately, were highest during the pre-monsoon period (March–May), irrespective of habitat, whereas densities were low in all cases during winter (December–February). The study indicates that season had the most significant effect on the relative abundance of house dust mites except Dermatophagoides farinae, irrespective of habitat.     

屋尘螨致敏/激发小鼠气道变态反应性炎症模型的构建

目的使用屋尘螨(HDM)提取液致敏和激发C57BL/6小鼠构建气道变态反应性炎症模型的方法。方法模型组和对照组,各8只。模型组以HDM致敏和激发小鼠,分别于第0、7、14、21天腹腔注射HDM,10 d后,连续雾化吸入HDM 3 d。对照组以PBS代替HDM。进行支气管肺泡灌洗液(BALF)细胞总数计数和分类计数,肺组织病理检查,ELISA测定BALF上清中IL-4、IL-5和IFNγ-水平。结果模型组可见明显炎性细胞浸润,以嗜酸性粒细胞为主。而对照组未见明显炎性细胞浸润。BALF中细胞总数计数和嗜酸性粒细胞比例较对照组明显升高(P<0.01);BALF上清中IL-4、IL-5水平较对照组明显升高(P<0.001),IFNγ-水平较对照组降低(P<0.01)。结论使用HDM致敏和激发C57BL/6小鼠成功地建立了小鼠气道变应性炎症模型。     

Temperature-dependent population growth of three species of stored product mites (Acari: Acaridida)

The pest potential of stored product mites depends on the reproduction rate that is affected by the environmental conditions. In this study we investigated the effect of temperature, ranging from 5 to 35°C, on the population growth of three important mite species, Acarus siro, Tyrophagus putrescentiae and Auleroglyphus ovatus at 85% r.h. Starting with 10 individuals the population increase of mites was observed after 3 weeks of cultivation, or after 6 weeks for those kept at low temperatures (5, 10, 12.5, and 15°C). The rate of increase was calculated for each temperature and species. The obtained data were fitted with polynomial models. The mite population growth rates increased with increasing moderate temperatures until 25°C, when r _m -values were 0.179, 0.177 and 0.190 for A. siro, A. ovatus and T. putrescentiae, respectively. The lower development threshold was 10.2°C in all three species. Estimated upper temperature threshold was higher in T. putrescentiae (49°C) than in A. siro and A. ovatus (38°C). Simulation of the rate of population increase under ideal conditions, using real temperature records obtained from Czech grain stores, showed that the pest mite populations increase only during 3.5 months within a typical 9-month storage season in Central Europe. These results indicate that control of mites, be it chemical, physical or biological, is recommended during the months when allergens and pests are produced, i.e. from September to mid November and in May.     

Contamination of Passenger Trains with Dermatophagoides (Acari: Pyroglyphidae) Mite Antigen in Japan

Passenger trains were surveyed for contamination with Dermatophagoides farinae Hughes and Dermatophagoides pteronyssinus (Trouesart) mites in Japan. A total of 492 dust samples were collected from upholstered seats in six commuter trains, one long-distance express train and three night trains in October, 1996 and January, April, and July, 1997. Mite antigen levels contained in fine dust fractions of these samples were measured by an enzyme- linked immunosorbent assay. Most samples obtained from commuter trains showed relatively high mite antigen levels of >10gm^–2 (corresponding to >100 mites). Express and night trains showed lower antigen levels per square meter, but higher mite antigen levels per gram of fine dust than commuter trains, indicating relatively high mite antigen densities. Seasonal comparisons indicated that commuter trains showed the highest mean antigen level per square meter in winter (January), whereas the highest antigen level per gram of fine dust was observed in summer (July) in express and night trains.     

Asthma induced by house dust mites: benefits resulting from a correct environmental hygienization

Summary The aim of the present work has been the determination of the effectiveness of a primary prevention in the allergopathy induced by house dust mites weighing its benefits as regards asthmatic symptomatology.A sample of 56 subjects, aged 6 to 30 years, monosensitive to house dust mites (DTP and DTF) and with a diagnosis of bronchial asthma has been examined. The patients have entered a study plan whose purpose has been the evaluation of the effects of an environmental improvement on symptomatology. The diagnosis of asthma induced by house dust mites has been confirmed both by in vivo (Prick-test) and in vitro (PRIST and RAST) investigations.A follow-up after 3 months has shown that the hygienic measures have produced beneficial effects on symptomatology in as many as 22 patients (52.4%) out of a total of 42 who have put into practice the environmental improvement. These results encourage to proceed on this path in the choice of the therapy for those subjects affected by respiratory allergopathy to house dust mites.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Abstract

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

Generation of a transgenic rice seed-based edible vaccine against house dust mite allergy

As an alternative approach to conventional allergen-specific immunotherapy, transgenic rice seed expressing a major house dust mite (HDM) allergen, Der p 1, was developed as an edible vaccine. The C-terminal KDEL-tagged Der p 1 allergen specifically accumulated in seed endosperm tissue under the control of the endosperm-specific GluB1 promoter. Der p 1 reached a maximum concentration of 58 μg/grain and was deposited in the endoplasmic reticulum (ER)-derived protein body I (PB-I). Plant-derived Der p 1 was posttranslationally modified with high-mannose-type glycan structures. Glycosylated Der p 1 displayed reduced IgE binding capacity in comparison with its unglycosylated counterpart in vitro. Our results indicate that transgenic Der p 1 rice seeds are a safe, potential oral delivery vaccine for the treatment of HDM allergy.     

尘螨变应原Der f 1核酸序列测定及其分子进化分析

目的：对尘螨主要变应原Der f 1进行核酸序列测定,探讨其系统进化信息。方法：根据Genbank公布的Der f 1基因序列设计引物,巢式PCR扩增Der f 1的cDNA,纯化、回收、克隆至pMD19-T simple后进行序列测定,序列比对后用Clustal W1.83构建分子进化树。结果：成功扩增出Der f 1的cDNA片段,测序表明该基因含ORF1个,长度966bp,与参考序列同源性达99．9％。该变应原具半胱氨酸蛋白酶活性,与果蝇进化关系最远,与梅氏嗜霉螨进化关系最近。结论：成功获得了尘螨变应原Der f 1基因片段．根据其编码的氨基酸序列构建出的系统进化树与形态学分类不一致。     

Phthalocyanine-dyed fibers adsorb allergenic proteins

Summary. Phthalocyanine (Pc)-dyed fiber is reported to reduce atopic symptoms in some patients when they use underwear made of the fiber. We investigated the adsorption of allergens on Pc-fiber. Pc-fiber trapped house dust/pollen/food allergens with varied molecular weight and pI. The adsorbed allergens were released in the presence of mild detergent. Pc-fiber did not change the molecular weight or disulfide bonding of the allergens. These observations imply that Pc-fiber is applicable as an “allergen trap” for a wide variety of products.     

High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells

Blo t 5 is a major allergen from house dust mite Blomia tropicalis. Purification of native Blo t 5 (nBlo t 5) from whole dust mite extract is tedious and gave low yield. In this study, we demonstrated that codon optimization facilitated high-level expression of Blo t 5 in Chinese hamster ovary (CHO)-K1 cells and thus allows production of sufficient recombinant cBlo t 5 for specific immunotherapy. A codon optimized Blo t 5 gene was synthesized by PCR and the codon optimized or wild-type Blo t 5 gene in pcDNA3.0 was transfected into CHO-K1 cells and stably selected with Geneticin (G418). Western-immunoblot analysis of spent culture media detected a positive band at 14kDa for the codon optimized but not wild-type gene transfectants. In addition, a stable CHO-K1 clone produced up to 13 mg/L of the cBlo t 5 protein having a high correlation of human IgE reactivities and allergenicity to the native Blo t 5, thus indicating proper conformation of this recombinant form.