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PubMed2003

Cloning and molecular characterization of a cDNA from Blomia tropicalis homologous to dust mite group 3 allergens (trypsin-like proteases).

BACKGROUND: Exposure to the house dust mite Blomia tropicalis is increasingly being implicated as a major risk factor for asthma exacerbations in sensitized individuals. The objective of this study is to clone and characterize B. Tropicalis allergens in order to better define their role in allergic asthma. METHODS: A lambda gt22A cDNA library was constructed from B. Tropicalis mRNA and screened using specific DNA probes. A full-length cDNA (Bt2-3) was isolated, subcloned, and sequenced. RESULTS: Sequence analysis showed that clone Bt2-3 encodes the full-length serine protease preproenzyme from B. Tropicalis. The predicted Bt2-3 protein consists of a 15-amino-acid signal peptide, a 20-amino-acid propeptide and a mature protein of 231 amino residues. BLAST analysis of the deduced amino acid sequence showed high homology (48-54%) between clone Bt2-3 and serine proteases (group 3 allergens) from domestic dust mites. Both the serine active site (DACQGDSGGPVA; amino acids 214-225) and the histidine active site (LTAAHC; amino acids 71-76) of serine proteases were highly conserved. The estimated molecular weight of the preproenzyme is 27.5 kD and its theoretical pI is 8.7. The mature protein contains a putative tyrosine kinase phosphorylation site (amino acids 24-31) and several N-myristoylation sites. Sequence alignment shows that the serine protease active sites are highly conserved among the clinically important mite species, including B. Tropicalis. CONCLUSION: We report the cloning and molecular characterization of a B. Tropicalis cDNA clone encoding a trypsin-like protease, with a possible major role as an allergen. Expression and further characterization of the recombinant product will help determine the role of proteolytic enzymes in the pathophysiology of allergic asthma.

Allergens↗
PubMed1982

Characterization of the carbohydrate moiety in a purified allergen preparation from the mite Dermatophagoides farinae and its importance for allergenic activity as tested by rast-inhibition method.

The monosaccharide composition of a purified mite allergen preparation was mainly found to be: mannose, galactose, glucose, N-acetylglucosamine and N-acetylgalactosamine quantitated to 15% of dry material by means of methanolysis and GLC of trimethylsilylated methyl glycosides. Protein content was found to be 65%, determined by the Lowry method. Methylation analysis and periodate oxidation indicated the main polysaccharide material to be built of 1,6-linked hexosamine, with side chains having mainly galactose end groups. Attempts to obtain separate carbohydrate fractions by ion exchange chromatography of the extract on CM-Sephadex and DEAE-Sephadex columns were not successful. The RAST-inhibition test showed a lower IgE binding capacity for periodate-treated mite preparation, where sugars containing vicinal diols have been oxidized, compared to an untreated mite preparation.

Allergens↗
PubMed1986

Detection of allergens in mould and mite preparations by a nitrocellulose electroblotting technique.

The allergen composition of moulds (Alternaria alternata and Aspergillus fumigatus) and mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) was studied by electroblotting. The allergens were separated by SDS-gPAGE and transferred to a nitrocellulose membrane by electroblotting. Specific IgE antibodies directed against mould and mite allergens were utilized as probes to detect the allergens. The bands were localized on the nitrocellulose membrane by means of enzyme- or isotope-labelled antibody. Circulating specific IgE antibodies in mould and mite sensitive patient sera were also analyzed and characterized with respect to their IgE specificity. Under appropriate experimental conditions this electroblotting technique could also be used for quantitation of individual allergens by scanning the isotope- or enzyme-stained nitrocellulose strips.

Allergens↗
PubMed1987

Percutaneous sensitisation to house dust mite may occur naturally in eczema.

The concentrations of serum IgE with specificity for the faecal and body allergens of the house dust mite, Dermatophagoides pteronyssinus, were compared in 69 children with various combinations of asthma, eczema and/or rhinitis. The concentration of IgE antibodies to the mite body was higher in those with eczema than those with asthma but the concentrations of anti-faecal antibodies were similar. Our results suggest that anti-mite body antibodies are characteristic of eczema and that sensitization to the body allergen may occur because of its passage through the skin.

Allergens↗
PubMed1987

In vitro translation of messenger RNA from the house dust mite Dermatophagoides pteronyssinus.

RNA was extracted from the house dust mite Dermatophagoides pteronyssinus and a polyadenylated [poly(A)+] and non-polyadenylated [poly(A)-] mRNA isolated by oligothymidylic acid-cellulose chromatography. Both preparations were translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of 35S-methionine into trichloroacetic acid-precipitable translation products obtained using poly(A)+ and poly(A)- mRNA was 21- and 1.3-fold, respectively, when compared with control translations performed without added mRNA. Sodium dodecylsulphate-polyacrylamide gel electrophoretic analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14,000- greater than 67,000 daltons were synthesized. Immunoprecipitation studies performed using a sheep anti-whole mite antiserum showed that several of the synthesized proteins corresponded with mite antigens. These findings were confirmed by coprecipitation studies using crossed immunoelectrophoresis (CIE). A comparison of the CIE data with those obtained with crossed radio-immunoelectrophoresis showed that 6 of the 9 allergens previously described in our laboratory were synthesized, namely Der p I, Dpt 1, 3, 4, 8 and 17. The allergenicity of the lysates was also confirmed by immunoprecipitation studies using a specific anti-human IgE reagent.

Allergens↗
PubMed1989

Strong delayed-type hypersensitivity induced against house dust mite antigens in the mice.

The aim of this study was to assess the antigenicity of house dust mite antigens using delayed-type hypersensitivity (DTH) responses in mice. The crude extracts of Dermatophagoides pteronyssinus and Dermatophagoides farinae induced strong DTH responses and was transferred with T cells. Assessment of DTH responses to the house dust revealed that substantial cross-reactivity at T-cell level was observed between these two mite extracts. Almost all the fractions of the extract of D. pteronyssinus, which has a molecular weight of between 10 and 100 kilodaltons, elicited strong DTH responses. It was found that antigenicity of the mite extract was reduced by chemical denaturation with tannic acid.

Allergens↗
PubMed1989

Characterization of the proteases in the crude mite extract.

Characterization of the proteases was performed in the crude mite extract fractionated by Sephacryl S-200 gel filtration. Three peaks of protease activities were detected in the fractions. From the results of substrate specificity and susceptibility to the inhibitors, PK.1 protease (about 60 kD) is suggested to be a trypsin-like protease of mites. From the results of susceptibility to various agents, PK.2 (about 30 kD) and PK.3 (about 20 kD) proteases may be cysteine proteases, e.g., papain and cathepsin B. PK.3 protease existed in the precipitate of 60% ammonium sulfate fractionation. The data in the present study suggest the possibility that Dermatophagoides farinae I allergen might be a cysteine protease probably derived from the gastrointestinal tract of the house dust mite.

Allergens↗
PubMed1990

Isolation of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay.

A lambda gt11 library made with cDNA from the house dust mite Dermatophagoides pteronyssinus was screened with human allergic serum by IgE plaque radioimmunoassay. This resulted in the isolation of clones coding for the major allergen Der p II. The cDNA coded for a 129-residue protein of 14,131 daltons with no N-glycosylation sites. No sequence homology with other proteins was evident. The Der p II expressed in Escherichia coli reacted with IgE in 14 of 17 sera from mite-allergic patients giving clonal evidence for its designation as a major allergen. This, along with previous work, has resulted in the cloning of the two major mite allergens.

Allergens↗
PubMed1994

Cloning and characterization of cDNA coding for a new allergen from the house dust mite, Dermatophagoides farinae.

A cDNA clone encoding a new allergen from the Dermatophagoides farinae cDNA lambda gt11 library was isolated and sequenced. There was no amino acid sequence homology with other known allergens. The gene product, beta-galactosidase fusion protein, of the truncated cDNA on blot reacted with IgE in 13 of 43 sera from patients allergic to mites. The affinity-purified fusion protein had a potent ability to release histamine from washed blood cells of the mite-allergic patients. Human specific IgE eluted from the fusion protein band on blots recognized a 39-kD component on blots of a mite body extract.

Allergens↗
PubMed1997

Genomic organization and polymorphisms of the major house dust mite allergen Der f2.

We amplified genomic DNA encoding the major house dust mite allergen, Der f2, from Dermatophagoides farinae by means of the polymerase chain reaction and cloned it into Escherichia coli. The nucleotide sequences of the amplified fragments were determined and compared with those of the cDNA previously reported. Four Der f2 genomic clones were obtained, suggesting that the genomic Der f2 gene had sequence polymorphisms like the cDNA clones. Each of the genomic clones had a single small intron. With respect to the exon sequences, two of the four genomic clones were identical with two cDNA clones, respectively. The others were combinations of the two clones. Genomic Southern blotting suggested that the Der f2 gene is located at one locus in the mite genome and that sequence substitutions were due to polymorphisms among individual mite genes.

Allergens↗
PubMed1997

Cyclic AMP-elevating agents inhibit mite-antigen-induced IL-4 and IL-13 release from basophil-enriched leukocyte preparation.

Human peripheral blood basophils are known to secrete interleukin (IL)-4 and IL-13 after cross-linking of cell surface IgE. However, little is known about the pharmacological regulation of allergic cytokine release from basophils. In the present study, we investigated the effects of cyclic 3',5'-adenosine monophosphate (cAMP)-elevating agents on antigen-induced IL-4 and IL-13 release from basophil-enriched leukocyte preparations. We obtained venous blood from 27 atopic asthmatic patients (mean age was 45.8+/-3.6 years, all patients were sensitive to mite antigen) and prepared basophil-enriched leukocyte preparations by double-Percoll gradients (basophil purity was 13.4+/-1.6%). The cell preparations were treated with phosphodiesterase (PDE) inhibitors, dexamethasone, forskolin or dibutyryl cAMP for 10 min and were challenged with mite antigen for 6 h. The released IL-4 and IL-13 in the supernatants were measured by enzyme-linked immunosorbent assay systems. No IL-4 or IL-13 was detected in the supernatant of the basophil-depleted preparation after the challenge with mite antigen, suggesting that basophils specifically produce these cytokines. A nonselective PDE inhibitor, theophylline, and a PDE IV-selective inhibitor, rolipram, significantly suppressed the release of IL-4 and IL-13 from the basophil-enriched preparation. Although several concentrations of cilostazol, a PDE III-selective inhibitor, had no effect on the release of both cytokines, cilostazol suppressed the release of IL-4 additively when applied with rolipram. Forskolin and dibutyril cAMP also significantly suppressed the release of both cytokines, suggesting that the suppressive effects by PDE inhibitors were accompanied by the elevations in cAMP levels. We conclude that basophil-enriched leukocyte preparations produce IL-4 and IL-13 in response to antigen and that the release of these cytokines could be regulated by cAMP-modulating agents.

Animals↗
PubMed1981

The distribution of dust mite allergen in the houses of patients with asthma.

Using an inhibition radioimmunoassay for the major allergen from Dermatophagoides pteronyssinus (antigen P1), we studied the distribution of this dust allergen in the houses of patients with asthma. Both bed and floor dust samples contained a wide range of antigen P1, 100 to 100,000 ng/g of fine dust, and this concentration correlated well with the number of mite bodies (r = 0.81, p less than 0.001). We were unable to detect antigen P1 in the air of undisturbed rooms. However, during domestic activity, between 1 and 30 ng were collected on a filter than sampled air for 45 min at 17 L/min. Using a cascade impactor it was shown that greater than 80% of the airborne antigen P1 was associated with particles greater than 10 mu in diameter. Some of the particles containing allergen could be identified because they formed precipitin rings when impacted onto agarose containing rabbit antimite antiserum. These particles had the physical appearance of mite feces, which are the major source of antigen P1 in mite cultures. The results suggested that natural exposure to this dust allergen allows occasional fecal particles to enter the lungs and that these particles contain very concentrated allergen.

Animals↗
PubMedDate not supplied

House dust mite-specific IgE, IgG1, and IgG4 antibodies in patients with perennial rhinitis.

Serum samples were obtained from patients with house dust mite-sensitive nasal allergies before and 6 months after immunotherapy, and the level of immunoglobulin (Ig)E, IgG1, and IgG4 antibodies in those sera was determined by the enzyme-linked immunosorbent assay. The present data showed that the mite-specific IgG4 antibodies increased in patients who responded well to immunotherapy but not in those who responded poorly. It was suggested from the present study that IgG4 antibodies seem to act as "blocking antibodies" to reduce the allergic reaction in the target organ, and that an increase of allergen-specific IgG4 antibodies following immunotherapy can be of clinical benefit to patients suffering from mite-specific nasal allergy.

Dust↗
PubMed1993

The same photoperiodic clock may control induction and maintenance of diapause in the spider mite Tetranchus urticae.

In the spider mite Tetranychus urticae, both diapause induction (which takes place during the larval and nymphal stages) and diapause maintenance (in the adult female) are under photoperiodic control. The question of whether or not the same photoperiodic clock is involved in both photoperiodic reactions was investigated in eight strains of the spider mite, originating from different localities in Europe. The methods employed consisted of (1) determination of the relative importance of the photophase and scotophase in the two photoperiodic reactions; (2) comparison of photoperiodic response curves for diapause induction and diapause maintenance; and (3) determination of the effect of light breaks on the capacity of long nights to maintain diapause, and comparison with the effect of light breaks in diapause induction experiments. The scotophase appeared to be much more important than the photophase for both diapause induction and diapause maintenance. In all strains the critical daylength for diapause maintenance, measured at the moment of saturation of the response to long daylengths, was identical to the critical daylength for diapause induction. However, the critical daylength for diapause maintenance appeared to be labile; it shifted gradually to shorter values as the mites were kept in the cold for a longer period of time, or were kept at a higher temperature for a progressively longer period of time after their stay in the cold room. This seems to reflect a gradual loss of photoperiodic control of diapause maintenance as diapause development proceeds. Photoperiods close to the critical daylength appeared to be less strong with regard to diapause maintenance than shorter daylengths. Quantitative differences in the "strength" of different daylengths were found in all strains investigated. Interruption of the night by short pulses of light revealed either one or two peaks of sensitivity in the night, or one broad "trough" where the two peaks had merged. However, in each case maximal sensitivity to the light breaks occurred at the same position in the night for diapause induction and diapause maintenance. The many similarities found lead to the conclusion that most probably the same photoperiodic clock mechanism is involved in both diapause induction and diapause maintenance in T. urticae.

Animals↗
PubMed1993

Decrease of skin and bronchial sensitization following short-intensive scheduled immunotherapy in mite-allergic asthma.

Changes induced by high-dose intensive specific immunotherapy (ISI) were studied in 18 patients with mite-allergic bronchial asthma and compared with 18 control patients. A biologically standardized well-characterized Dermatophagoides pteronyssinus extract of known potency was used both for in vivo tests and ISI. Immediate D pteronyssinus skin tests (measured by parallel-line bioassay) were reduced 20-fold in ISI treated patients, and delayed skin responses significantly decreased and even disappeared following ISI. Bronchial tolerance to D pteronyssinus increased an average of ten times in ISI-treated patients, whereas nonspecific hyperreactivity remained unchanged. ISI induced significant specific IgG and IgG4 increases but no changes were observed in specific IgG1,2 and 3 or in specific IgE. Clinical score did not change significantly after ISI, short ISI schedule with a well-characterized mite extract greatly reduced the degree of mite sensitization (skin and bronchial allergic responses), but clinical disease failed to improve significantly, probably owing to the lack of influence of ISI in nonspecific hyperreactivity.

Adolescent↗
PubMed2006

Relationships between the parasitic mite Spinturnix andegavinus (Acari: Spinturnicidae) and its bat host, Myotis daubentonii (Chiroptera: Vespertilionidae): seasonal, sex- and age-related variation in infestation and possible impact of the parasite on the host condition and roosting behaviour.

Host-parasite relationships between the Daubenton's bat, Myotis daubentonii Kuhl, 1817 (Chiroptera: Vespertilionidae), and its haematophagous ectoparasite, the mite Spinturnix andegavinus Kolenati, 1857 (Acari: Spintumicidae), were subjected to analyses based on data gathered during a six-year study (1999-2004) within a single study area in South Bohemia, Czech Republic. Seven hundred and fifty-one Daubenton's bats were examined by screening wing membranes with an intensive light source, resulting in 4,690 recorded mites. Sex, age, weight and reproductive state were evaluated for each bat. A body condition index was calculated as a ratio of weight to forearm length. The seasonal course of mite infestation displayed distinct dynamics with the peak during the lactation and post-lactation periods coinciding with occurrence of the most numerous colonies of Daubenton's bats in the study area. Infestation rates differed between the two sexes, being higher in adult females than adult males. Juvenile bats of both sexes (with no differences between males and females) were the most infested group of all. Pregnant females had a significantly higher parasite load than non-pregnant ones, while no differences in infestation rates were found between lactating and non-lactating females. The analyses of the relationship between parasite load and body condition of bats revealed no common trends for all sex- and age-related groups. Two possible explanations are suggested and discussed: (1) There is no true relationship between the two tested variables and, thus, the significant results were attained due to a random statistical effect. (2) Different underlying causal mechanisms may exist that influence parasite load and, especially, body condition, with respect to the particular sex and age category of bats. The seasonal roosting dynamics of the Daubenton's bat are suggested to be the result not only of changing energetic demands of resident population members, but also of coevolutionary strategies within host-parasite relationships.

Acari↗
PubMed2001

House dust mites in Brazil--an annotated bibliography.

House dust mites have been reported to be the most important allergen in human dwellings. Several articles had already shown the presence of different mite species at homes in Brazil, being Pyroglyphidae, Glycyphagidae and Cheyletidae the most important families found. This paper is an annotated bibliography that will lead to a better knowledge of house dust mite fauna in Brazil.

Animals↗
PubMed2003

Infestation and distribution of the mite Varroa destructor in colonies of Africanized bees.

Whereas in several parts of the world varroa is the major pest affecting apiculture, in others the parasite is unknown to many beekeepers because its damage to bees is minor. The impact of the mite Varroa destructor is related to the climatic conditions and the races of Apis mellifera bees in each region where the pest exists. In the present study, the current level of infestation by the mite was assessed to determine the evolution of the pest in Africanized bee colonies in Southern Brazil. This level of infestation was considered low: approximately two mites per one hundred adult bees. This result is similar to that obtained for the same apiary almost five years ago and for others distributed in various regions of Brazil. In the present study, we also estimated the total varroa population and its distribution among brood and adults in each bee colony.

Animals↗