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.     

Quantitative determination of Der p I allergen levels in allergenic extracts and house-dust samples

Summary House-dust mites are responsible for serious respiratory diseases in humans. An ELISA assay to detectDermatophagoides pteronyssinus (Dp) mites has been set up. This assay, based on quantitative determination of the majorDp allergen, called Der p I, has been applied for the analysis of growing mite cultures and house-dust samples.Der p I allergen levels in mite cultures are well correlated with the number of mites present as well as with the biological activity of the corresponding extracts. Different methods for detectingDp mites in house-dust samples were compared. The ELISA method shows good sensitivity and specificity, and is particularly suitable for routine analyses.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

Evaluation of the immunospecific activity of allergens prepared from house dust and the mite Dermatophagoides pteronyssinus in the bacteriosorption reaction on a flat surface

The content of protein A-reactive IgG to allergens prepared from house dust and D. pteronyssinus was determined in the sera of 4 immunized rabbits, 10 sensitized and hyposensitized guinea pigs and 37 treated and untreated allergic patients by means of the previously developed bacteriosorption test (BST). The sensitivity of the test for the determination of allergen-specific antibodies was 50-100 ng/ml. This test was shown to permit the detection of IgG in the sera of immunized, sensitized and hyposensitized animals, as well as in the sera of some treated and untreated patients. The antigenic similarity of both allergens was confirmed. Three batches of D. pteronyssinus allergen, standardized as to the content of protein nitrogen, differed in their immunospecific activity in BST with one of the rabbit sera and with the set of the patients' sera. The covalent immobilization of house-dust allergen on polystyrene by means of water-soluble carbodiimide gave no advantages in BST in comparison with adsorption immobilization in alkaline carbonate-bicarbonate buffer.     

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.     

Identification of house-dust mites

The term house-dust mite usually refers to those species of the mite family Pyroglyphidae, that are known to commonly occur, although sometimes regionally, in the dust of human dwellings. These species belong to five genera:Dermatophagoides, Euroglyphus, Hirstia, andMalayoglyphus. Related species ofDermatophagoides have the most world-wide occurrence, the commonest being:D. farinae, D. microceras, andD. pteronyssinus. A correct taxonomic identification of house-dust mites is very important, not only from a biological stand point, but also regarding the consequences of their respective allergenical properties. Several immuno-chemical studies revealed differences between the products of two hard to distinguish sibling species. A preliminary practical taxonomic key for the most common and important house-dust mites is presented.     

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.     

Optimal efficacy of a fungicide preparation,natamycin, in the control of the house-dust mite,Dermatophagoides pteronyssinus

The efficacy of a fungicidal preparation, natamycin, for the effective control ofDermatophagoides pteronyssinus (Trouessart), was determined at different concentrations on mattress-dust microsites, including ticking. One treatment with the commercially available natamycin concentration (Tymasil) resulted in a 65% reduction of house-dust mite populations within 4 weeks of treatment. The acaricidal control could be proven despite the protective role of mattress fibres such as ticking. However, mite fecundity was much more affected after treatment with twice the commercial concentration, which then assured a long-term control. Concentrations below the commercially available one were not effective. In a maritime climate, six sequential treatments three times a year would be effective for an adequate long-term control because of the high growth of house-dust fungi and the close interaction between the mite and the mouldAspergillus penicilloides.     

Ecological relationships between xerophilic fungi and house-dust mites (Acarida: Pyroglyphidae)

Summary At. 75 and 80% relative humidity (RH), on a wheat germ flake medium, Aspergillus penicilloides grew abundantly and suppressed the population growth of Dermatophagoides pteronyssiunus. At 71% RH, A. penicilloides grew moderately and was only antagonistic to D. pteronyssinus when the fungus was previously incubated on the medium.On a human dander medium and on mattress dust, A. penicilloides grew moderately at 71% and 75% RH and stimulated the development of D. pteronyssinus populations. Also a moderate growth of Eurotium repens on human dander positively influenced D. pteronyssinus. Wallemia sebi and Penicillium brevicompactum grew slightly or did not grow at all at 75% RH. No effect was observed on D. pteronyssinus.It appears that xerophilic fungi may stimulate, and occasionally may reduce, the growth of house-dust mite populations in the natural environment.     

Effects of five insect growth regulators on laboratory populations of the North American house-dust mite,Dermatophagoides farinae

The potential of insect growth regulators (methoprene, hydroprene, fenoxycarb, diflubenzuron and triflumuron) to control populations of the North American house-dust miteDermatophagoides farinae (Hughes) was assessed in laboratory bioassays. Methoprene was most effective at suppressing population growth, especially at concentrations of 1.0% (10 000 ppm) and 5.0% (5000 ppm) active ingredient. Hydroprene, structurally related to methoprene, also suppressed house-dust mite populations but not as consistently as methoprene. Fenoxycarb may be effective at controlling house-dust mites but at greater concentrations than were tested. Diflubenzuron and triflumuron, two chitin-synthesis inhibitors, failed to suppress mite numbers and may, in fact, stimulate reproduction in some cases. Almost all concentration of the insect growth regulators were shown to be ineffective when assayed 90 days after treatment.     

A comparative study of allergenic and potentially allergenic enzymes fromDermatophagoides pteronyssinus,D. farinae andEuroglyphus maynei

The presence of the enzymatically active allergens equivalent toDer p I (cysteine protease),Der p III (serine protease) and amylase in extracts ofDermatophagoides pteronyssinus, D. farinae andEuroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived fromD. pteronyssinus andD. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared fromD. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Isolation and characterisation of a 13.8-kDa bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests that the enzyme could be bacterially derived

Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract. Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment. Substrate SDS-PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D. pteronyssinus spent growth medium extract by hydroxyapatite chromatography. The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies. The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence. The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily. These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se.     

Hyposensitization with Dermatophagoides pteronyssinus Antigen: Trial in Asthma Induced by House Dust

A double-blind clinical trial of hyposensitization with aqueous extracts of Dermatophagoides pteronyssinus (the house-dust mite) and human skin scales showed a substantial improvement in symptoms in 11 asthmatics allergic to house dust treated with the D. pteronyssinus extract and a reduction in their need for other therapy. Five patients were well for a year but six relapsed. These results contrasted with the generally unfavourable course of the patients treated with the extract of human skin scales. Asthma due to house-dust allergy may be substantially improved by hyposensitization with D. pteronyssinus extract.     

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.     

Air cleaners can reduce the risk of allergic sensitization

Exposure to house-dust mite allergens is a factor in the development of allergic symptoms in atopic patients. Several measures can be proposed to control indoor allergen levels, inducing a clinical benefit. The use of an air cleaner is one simple way of achieving this goal. We conducted a simulation trial in a proper room, to verify the usefulness of a domestic cleaner, based on mechanical air filtration, to reduce the levels of environmental allergens. We checked the presence of mite components by different methods (Aclotest and Der p 1 ELISA), in dust recovered before and after using the air cleaner. Our results indicate that this approach could be useful in significantly lowering the levels of mite allergens.     

Protein chips for detection of mite allergens using Kunitz-type protease inhibitors

Stored-food and house-dust arthropods include many species of mites and beetles that affect human health. For diagnostic tests proteases such as trypsin are utilized as they are indicators of the presence of allergen contaminants in food. We recently characterized Kunitz-type protease inhibitors (KPIs) from Solanum palustre. Here we studied biotechnological applications of KPI-B1 and -B4. We manufactured a protein chip with immobilized KPI-B1 and -B4 and showed trypsin/chymotrypsin-binding specificity, indicating that the recombinant proteins have protease selectivity. We employed the protein chip to capture mite proteins belonging to the protease family with polyclonal anti-mite antibodies. The mite diagnostic chip can be useful for detecting mite allergens.     

Mites in house dust and human allergoses

The literary data on the study of the house-dust mites during the period from 1864 till 1983 are given. The methods of the house dust samples collection in flats and the methods of the house-dust mites isolation from the house dust are evaluated. The house-dust mites fauna including more than 130 species is described. The synantropic mites-pyroglyphides are the main part of the fauna, the stored products mites and predatory cheyletid and gamasid mites occur regularly. The rest part of the fauna consists of the different mites got into house by chance. The literary data on the influence of temperature and relative humidity on the development and growth of the mite population are given. The house-dust mites feeding and digestion processes bearing relation to the formation of the mite allergen are reviewed. The original data complete the literary data on the fauna and distribution of the house-dust mites in Moscow.     

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.