Use of isoelectric focusing in studying the composition of allergens from mites

The method of the isoelectric separation of allergens isolated from Dermatophagoides ticks with the use of isoelectrofocusing has been improved. 10-12 protein fractions with isoelectric points from 3.50 to 6.55 have been isolated. Allergens obtained from D. farinae and D. pteronyssinus grown on two different nutrient media have been found to contain common protein components. In D. pteronyssinus allergen the presence of a greater number of protein fractions has been noted in the region of pH 5.25-6.00 than in D. farinae allergen.     

The comparative characteristics of different forms of allergens from mites of the genus Dermatophagoides]

The comparative study of allergenic preparations of Dermatophagoides mites [correction of ticks] from different manufacturers and the international standard preparations of such allergens was made. D. pteronyssinus and D. farinae allergens from the USSR and the Netherlands were studied. The samples under study were evaluated in a complex of in vivo and in vitro experiments. The preparations produced in the USSR and in the Netherlands exhibited pronounced specificity, but the allergens of both species from the Netherlands were more active. Some differences in the immunochemical characteristics of the preparations were noted.     

In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.     

Clinical Significance of Skin Reactions to Mite Extracts in Children with Asthma

The mite Dermatophagoides pteronyssinus has been identified in dust from the houses of children in Birmingham suffering from asthma. Skin tests were carried out on 150 asthmatic children with extracts of D. pteronyssinus, of the related species D. farinae, of other mites found in house dust, and of crude house dust. Though positive reactions to D. pteronyssinus were obtained more frequently and were of greater size than those to the other extracts, it was considered that D. farinae is a suitable substitute for D. pteronyssinus for skin testing.In further tests on 302 asthmatic children with mite extracts and with extracts of allergens obtained commercially reactions to the former extracts were much more common than reactions to the latter.Major skin reactions (weals with diameter of 5 mm. or more) were present in 77% of children with a history of perennial asthma and house-dust sensitivity. Hence allergy to house-dust mites, particularly D. pteronyssinus, is of considerable importance in childhood asthma, and further study of the ecology and control of the mites in dust is desirable.     

Methods of detection of tick allergens in house dust

Different detection methods of housing dust allergens (belonging to the tick species Dermatophagoides pteronyssinus, D. farinae) were considered. As shown in this study, the immunochemical method in combination with acarological analysis proved to be most promising. On the basis of the data on the cross activity of tick allergens and changes in their content in dust, grounds were given for the expediency of detection by the immunochemical method with the use of polyclonal antibodies to specially obtained immunogen.     

Antigen Der f I from the dust mite Dermatophagoides farinae: structural comparison with Der p I from Dermatophagoides pteronyssinus and epitope specificity of murine IgG and human IgE antibodies

The physicochemical and antigenic properties of an allergen purified from Dermatophagoides farinae, Der f I, were compared with Der p I from Dermatophagoides pteronyssinus. On SDS-PAGE, Der f I migrated as a single polypeptide chain with the same m.w. as Der p I (24,000). Two isoallergenic peaks of Der f I were identified on preparative isoelectric focusing (pI 5.7 to 6.3 and pI 6.6 to 6.95). Fractions from each peak were shown to have an identical amino acid composition (which was similar but not identical to Der p I) and the same N-terminal amino acid sequence. There was a good correlation between quantitative intradermal skin tests to both purified allergens and to D. farinae extract in mite-allergic patients, with positive results when using as little as 10(-5) micrograms/ml of Der f I. The majority of sera with detectable IgE antibody to D. farinae also had IgE antibody to Der f I both among children (29/42 = 69%) and adults (55/63 = 87%). By RAST, there was an excellent correlation between IgE antibody to Der f I and Der p I in sera from 42 mite-allergic children (n = 0.94, p less than 0.001). Polyclonal IgG antibodies from six mice immunized with Der f I showed preferential binding to that allergen, and most monoclonal antibodies (16 of 18) raised against Der f I did not bind Der p I. However, two monoclonal antibodies from this fusion showed cross-reactive binding to both allergens. Immunoabsorption experiments, using D. pteronyssinus and D. farinae extracts coupled to Sepharose, showed that a large proportion of murine antibodies (74% to Der p I and 60 to 93% to Der f I) could not be absorbed by the heterologous extract on the immunosorbent. In contrast, in sera from seven mite-allergic patients, most of the specific IgE and IgG antibody (i.e., greater than or equal to 82%) was removed by either immunosorbent. Thus, Der f I and Der p I represent a homologous pair of major allergens which possess both cross-reacting and species-specific epitopes. The antibody response in mice immunized with either allergen in complete Freund's adjuvant was largely directed against species-specific epitopes, whereas in allergic humans, IgE- and IgG-specific antibodies bound predominately to cross-reacting epitopes.     

Major house dust mite allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 degrade and inactivate lung surfactant proteins A and D

Lung surfactant proteins (SP) A and D are calcium-dependent carbohydrate-binding proteins. In addition to playing multiple roles in innate immune defense such as bacterial aggregation and modulation of leukocyte function, SP-A and SP-D have also been implicated in the allergic response. They interact with a wide range of inhaled allergens, competing with their binding to cell-sequestered IgE resulting in inhibition of mast cell degranulation, and exogenous administration of SP-A and SP-D diminishes allergic hypersensitivity in vivo. House dust mite allergens are a major cause of allergic asthma in the western world, and here we confirm the interaction of SP-A and SP-D with two major mite allergens, Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1, and show that the cysteine protease activity of these allergens results in the degradation of SP-A and SP-D under physiological conditions, with multiple sites of cleavage. A recombinant fragment of SP-D that is effective in diminishing allergic hypersensitivity in mouse models of dust mite allergy was more susceptible to degradation than the native full-length protein. Degradation was enhanced in the absence of calcium, with different sites of cleavage, indicating that the calcium associated with SP-A and SP-D influences accessibility to the allergens. Degradation of SP-A and SP-D was associated with diminished binding to carbohydrates and to D. pteronyssinus 1 itself and diminished capacity to agglutinate bacteria. Thus, the degradation and consequent inactivation of SP-A and SP-D may be a novel mechanism to account for the potent allergenicity of these common dust mite allergens.     

Allergenic mites (Acariformes, Pyroglyphidae) in house dust

Paper is the second part of the literature review on house dust mites (including 1985). It deals with the diagnosis of the dust mite allergy, the nature of the mite allergens, distribution and population density of the mites in various premises, seasonal population dynamics and population age structure of the dermatophagoid mites, life cycle and breeding of Dermatophagoides pteronyssinus and D. farinae on different food substrates. The methods of the house dust mite control are discussed. The original data on the house dust mite distribution in Moscow is shown. A comparative estimation of the results of mite antigens in the house dust discovering by the acarological and three immunological methods are given.     

山西省晋中市及周边地区螨虫分布状况和优势螨种的研究

目的 研究山西省晋中市及周边地区的尘螨种类和优势螨种.方法 以就诊于晋中市第一人民医院耳鼻喉科门诊的尘螨过敏患者为研究对象,从2009年12月至2011年10月对居住在晋中市及周边地区的30户家庭进行入户采集调查.采集尘样后经过分离,制片,最后进行鉴定.结果 共采集尘样196份,户尘螨占成螨总数的60.1％,粉尘螨占39.8％.通过对不同采集地点的调查结果显示,楼房的样本阳性率为35.8％,平房的样本阳性率为21.8％.通过对不同环境样本中螨密度的统计,枕头的螨密度最高,为166.7头/g.平均螨密度在全年有2个高峰期,分别是7-8月份和12～2月.结论 晋中市及周边地区的主要螨种为麦食螨科的户尘螨和粉尘螨,户尘螨为优势螨种.     

House dust mite allergens in domestic homes in Cheonan, Korea

House dust mites produce inhalant allergens of importance to allergic patients. We measured the major group 1 allergens, Der p 1 and Der f 1, from the house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farina, respectively in 100 randomly selected domestic homes from Cheonan, Korea. Dust samples were collected by vacuuming from the living room floor and 1 mattress in each home. Der p 1 and Der f 1 were measured by double monoclonal ELISA. Der p 1 levels were very low, with geometric mean levels for floors and mattresses being 0.11 microgram/g (range: 0.01-4.05) and 0.14 microgram/g (range: 0.01-30.0), respectively. Corresponding levels of Der f 1 were higher, 7.46 microgram/g (range: 0.01-262.9) and 10.2 microgram/g (range: 0.01-230.9) for floors and mattresses, respectively. D. farinae appears to be the dominant house dust mite in Cheonan.     

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。     

Acaricidal effects of quinone and its congeners and color alteration of Dermatophagoides spp. with quinone

Acaricidal activity of the active constituent derived from Pyrus ussuriensis fruits against Dermatophagoides farinae and D. pteronyssinus was examined and compared with that of the commercial benzyl benzoate. The LD50 value of the ethyl acetate fraction obtained from the aqueous extract of P ussuriensis fruits was 9.51 and 8.59 microg/cm3 against D. farinae and D. pteronyssinus, respectively. The active constituent was identified as quinone by spectroscopic analyses. On the basis of LD50 values with quinone and its congeners, the compound most toxic against D. farinae was quinone (1.19 microg/cm3), followed by quinaldine (1.46), benzyl benzoate (9.32), 4-quinolinol (86.55), quinine (89.16), and 2-quinolinol (91.13). Against D. pteronyssinus, these were quinone (1.02 microg/ cm3), followed by quinaldine (1.29), benzyl benzoate (8.54), 4-quinolinol (78.63), quinine (82.33), and 2-quinolinol (86.24). These results indicate that the acaricidal activity of the aqueous extracts can be mostly attributed to quinone. Quinone was about 7.8 and 8.4 times more toxic than benzyl benzoate against D. farinae and D. pteronyssinus. Additionally, quinaldine was about 6.4 and 6.6 times more toxic than benzyl benzoate against D. farinae and D. pteronyssinus, respectively. Furthermore, the skin color of the dust mites was changed from colorless-transparent to dark brown-black by the treatment of quinone. These results indicate that quinone can be very useful as potential control agents, lead compounds, or the indicator of house dust mites.     

Prevalence of arthropod antibodies in Korean patients with allergic rhinitis.

Arthropod antigens are main causative agents which induce allergic responses in humans. However, little information is known about the prevalence of specific arthropod allergens in Koreans with allergic diseases. The current study was designed to determine the positive rates of arthropod antibodies by the Korean inhalant panel of MAST-CLA. One hundred sixty patients, who were diagnosed with allergic rhinitis from an out-patient center at the Soonchunhyang University Chunan Hospital, were studied between August 1998 to July 2000. The overall positive rate, at least more than one specific antibody of arthropods such as Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dp), and cockroach mix (Cm), was 46.9%. Each positive rate of Df, Dp, and Cm was 45.0%, 43.1%, and 8.8%, respectively. A significant agreement among arthropod allergens was observed (Df and Dp: 95.6%, Kappa = 0.911, P < 0.001). Our data supported the fact that arthropods were the most common allergens in Korean patients with allergic rhinitis; however, the MAST-CLA should be modified to increase specificity of arthropod allergens.     

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.     

Detection of mite antigens in house dust in Moscow

The acarological+ study of dust samples from the homes of allergic patients and normal persons in Moscow mite antigens were detected, respectively, in 66.7% and 38.1% of homes. The indirect mast cell degranulation test and the brain gliacyte volume change test in white rats gave similar results. The occurrence of Dermatophagoides pteronyssinus was approximately twice as great of D. farinae, and the numerical prevalence of the former species over the latter one was 1.3-fold. Dermatophagoides mites occurred in the homes of allergic patients 1.8-2.3 times as frequently as in the homes of normal persons.     

Mite-control activities of active constituents isolated from Pelargonium graveolens against house dust mites

The mite-control activities of materials obtained from Pelargonium graveolens oil against Dermatophagoides farinae and D. pteronyssinus were examined using an impregnated fabric disk bioassay and were compared with those shown by commercial benzyl benzoate and N,N-diethylm- toluamide (DEET). Purification of the biologically active constituents from P. graveolens oil was done by silica gel chromatography and high performance liquid chromatography. The structures of the active components were analyzed by EI/MS, (1)H-NMR, (13)C-NMR, (1)H-(13)C COSYNMR, and DEPT-NMR spectra, and were identified as geraniol (C(10)H(18)O, MW 154.25, trans-3,7-dimethyl-2,6- octadien-1-ol) and beta-citronellol (C(10)H(20)O, MW 156.27, 3,7-dimethyl-6-octen-1-ol). Based on the LD50 values, the most toxic compound was geraniol (0.26 microg/cm(2)), followed by beta-citronellol (0.28 microg/cm(2)), benzyl benzoate (10.03 microg/ cm(2)), and DEET (37.12 microg/cm(2)) against D. farinae. In the case of D. pteronyssinus, geraniol (0.28 microg/cm(2)) was the most toxic, followed by beta-citronellol (0.29 microg/cm(2)), benzyl benzoate (9.58 microg/cm(2)), and DEET (18.23 microg/cm(2)). These results suggest that D. farinae and D. pteronyssinus may be controlled more effectively by the application of geraniol and beta-citronellol than benzyl benzoate and DEET. Furthermore, geraniol and beta-citronellol isolated from P. graveolens could be useful for managing populations of D. farinae and D. pteronyssinus.     

Allergenic activity of mites of the genus Dermatophagoides

The allergenic activities of the laboratory batches of D. farinae allergens have been studied by the methods of indirect mast-cell degranulation, neuroglial cytocrit, electrophoretic mobility changes. D. farinae allergens have been shown to possess specific activity. The method of changes in the electrophoretic mobility of sheep red blood cells has demonstrated that D. pteronyssimus and D. farinae allergens possess common allergenic properties.     

Immunotherapy in the complex treatment of patients with atopic dermatitis with sensitization to house dust mites

Ninety-nine patients aged 16-52 years with disseminated atopic dermatitis at the remission were examined and treated. The patients were divided into 3 groups according to the method of therapy. Group 1 (28 patients aged 17-52 years, found to be sensitive to Dermatophagoides pteronyssinus and D. farinae) received, in addition to standard treatment, immunotherapy (oral hyposensitization with mite allergens) in combination with ointment containing retinopalmitate and methyluracil. Group 2 (39 patients aged 17-40 years) received standard treatment combined with the administration of placebo. Group 3 (32 patients aged 16-27 years) received only standard therapy. Groups 2 and 3 were used for comparison. The results of treatment were evaluated according to changes in the immune status of the patients and a complex of clinical characteristics. Essential improvement in clinical characteristics and the normalization of immunological parameters were registered in group 1, which proved that immunotherapy was effective and safe.     

Acaricidal activity and function of mite indicator using plumbagin and its derivatives isolated from Diospyros kaki Thunb. roots (Ebenaceae)

Acaricidal effects of materials derived from Diospyros kaki roots against Dermatophagoides farinae and D. pteronyssinus were assessed using impregnated fabric disk bioassay and compared with that of the commercial benzyl benzoate. The observed responses varied according to dosage and mite species. The LD50 values of the chloroform extract of Diospyros kaki roots were 1.66 and 0.96 microg/cm2 against D. farinae and D. pteronyssinus. The chloroform extract of Diospyros kaki roots was approximately 15.2 more toxic than benzyl benzoate against D. farinae, and 7.6 times more toxic against D. pteronyssinus. Purification of the biologically active constituent from D. kaki roots was done by using silica gel chromatography and high-performance liquid chromatography. The structure of the acaricidal component was analyzed by GCMS, 1H-NMR, 13C-NMR, 1H-13C COSY-NMR, and DEPTNMR spectra, and identified as plumbagin. The acaricidal activity of plumbagin and its derivatives (naphthazarin, dichlon, 2,3-dibromo-1,4-naphthoquinone, and 2-bromo-1,4- naphthoquinone) was examined. On the basis of LD50 values, the most toxic compound against D. farinae was naphthazarin (0.011 microg/cm2) followed by plumbagin (0.019 microg/cm2), 2- bromo-1,4-naphthoquinone (0.079 microg/cm2), dichlon (0.422 microg/ cm2), and benzyl benzoate (9.14 microg/cm2). Additionally, the skin color of the dust mites was changed from colorless-transparent to dark brown-black by the treatment of plumbagin. Similar results have been exhibited in its derivatives (naphthazarin, dichlon, and 2-bromo-1,4-naphthoquinone). In contrast, little or no discoloration was observed for benzyl benzoate. From this point of view, plumbagin and its derivatives can be very useful for the potential control agents, lead compounds, and indicator of house dust mites.     

Validity of MAST-CLA for diagnosis of arthropod allergy using receiver operating characteristic (ROC) analysis

Many allergists are currently focusing on the development of new diagnostic tools, and are attempting to improve both the sensitivity and specificity. A multiple allergen simultaneous test-chemiluminescent assay (MASTCLA) is one of the most popular diagnostic tools used in the Republic of Korea. However, there remains controversy among allergists with regard to the cut-off point for a positive result. The present study was conducted in order to determine the validity of MAST-CLA as compared with that of the skin prick test, with particular emphasis on arthropod allergens, on the basis of percentage agreement rates and kappa-values, and also to suggest the optimal positive cutoff points using receiver operating characteristic (ROC) curves. The study was conducted with 97 subjects (54 men, 43 women). Optimal individual cut-off points were calculated as follows; class II for Dermatophagoides farinae, class I for Dermatophagoides pteronyssinus, and trace for a cockroach mix. These findings suggest that attempting to apply optimal individual cut-off points will be a good way of improving diagnostic tests, particularly MAST-CLA.