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.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

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.     

The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.     

The crystal structure of a major dust mite allergen Der p 2, and its biological implications

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.     

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.     

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.     

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。     

Predicting the population dynamics of the house dust mite Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) in response to a constant hygrothermal environment using a model of the mite life cycle

A generalised model of the life cycle of a house dust mite, which can be tailored to any particular species of domestic mite, is presented. The model takes into account the effects of hygrothermal conditions on each life cycle phase. It is used in a computer simulation program, called POPMITE, which, by incorporating a population age structure, is able to predict population dynamics. The POPMITE simulation is adapted to the Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) (DP) mite using published data on the egg development period, total development period, adult longevity, mortality during egg development, mortality during juvenile development, and fecundity of individual DP mites held at a range of constant hygrothermal conditions. An example is given which illustrates how the model functions under constant hygrothermal conditions. A preliminary validation of POPMITE is made by a comparison of the POPMITE predictions with published measurements of population growth of DP mites held at a range constant hygrothermal conditions for 21 days. The POPMITE simulation is used to provide predictions of population growth or decline for a wide range of constant relative humidity and temperature combinations for 30 and 60 days. The adaptation of the model to correctly take account of fluctuating hygrothermal conditions is discussed.     

Unlocking the allergenic structure of the major house dust mite allergen der f 2 by elimination of key intramolecular interactions

We report on the structural background of the remarkable reduction of allergenicity in engineering of the major house dust mite allergen Der f 2. Disruption of intramolecular disulfide bonds in Der f 2 caused extensive conformational change that was monitored by circular dichroism and gel-filtration analysis. The degree of conformational change correlated well with the degree of reductions in the capacity to bind IgE and to induce histamine release from basophils in mite-allergic patients. Loosening the rigid tertiary structure by elimination of key intramolecular interactions is an effective strategy to reduce the number of high affinity IgE epitopes of allergen vaccine.     

Nuclear magnetic resonance structure and IgE epitopes of Blo t 5, a major dust mite allergen

A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.     

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。     

Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.     

Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity.

Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino-acid residues. Wild-type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel-filtration were 14-15 kDa. The IgE-binding capacity in the sera of seven mite-allergic patients, inhibitory activity for IgE-binding to immobilized wild-type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild-type Der f 2 in their CD spectrum, showed reduced IgE-binding, reduced inhibitory activity, and less histamine-releasing activity than the wild-type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE-binding site common to all tested patients. P66A showed reduced IgE-binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE-binding while the other mutants showed IgE-binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy.     

Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting

A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli. The cells were solubilized and run on SDS-PAGE under reducing conditions. Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.     

Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA.

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides. A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 micrograms/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.     

Effects of the fungus Aspergillus penicillioides on the house dust mite Dermatophagoides pteronyss'mus: an experimental re-evaluation

Abstract. In this report the widely-held view that house dust mites benefit from fungal contamination of the dietary substratum is re-examined. The performance of Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) is documented over two successive generations in the presence or absence of the xerophilic fungus Aspergillus penicillioides (Hyphomycetales: Moniliaceae). This fungus reduced survival, development rate, adult length and fecundity of D.pteronyssinus. Detrimental effects of A.penicillioides were proportional to the fungal density. Despite the antagonistic effects of A.penicillioides, a requirement for the fungus was indicated by the poor performance of fungus-free mites in the second generation; sustained culture of D.pteronyssinus in the absence of fungi is probably not possible. It is suggested that fungi may alter the particulate nature of the substratum to the detriment of house dust mites, but also provide micronutrients deficient in the diet.