Molecular-based allergy diagnosis of allergic bronchopulmonary aspergillosis in Aspergillus fumigatus-sensitized Japanese patients.

BACKGROUND: Distinguishing between patients with allergic bronchopulmonary aspergillosis (ABPA) and Aspergillus fumigatus (Af)-sensitized asthmatic patients without ABPA is sometimes difficult owing to the IgE-cross-reactivity between Af and other fungal allergens. OBJECTIVE: To establish the usefulness of molecular-based allergy diagnostics using allergen components from Af in distinguishing ABPA from Af-sensitized asthma without ABPA. METHODS: Sera from Japanese patients with ABPA (n = 53) and Af-sensitized asthma without ABPA (n = 253) were studied. The levels of IgE and IgG antibodies to allergen components from Af and IgE antibodies to different fugal allergen extracts were measured by ImmunoCAP. Comorbid atopic dermatitis (AD) was taken into consideration in the sensitization profile analysis. RESULTS: Patients with ABPA possessed significantly higher levels of IgE antibodies to Asp f 1, and Asp f 2 than asthmatic patients without ABPA. The areas under the receiver operating characteristic curves for the levels of IgE to Asp f 1 and Asp f 2 as diagnostic markers of ABPA were 0.75 and 0.78, respectively. The presence of IgE positivity to Asp f 1 and/or Asp f 2 resulted in increased sensitivity while losing little specificity. Comorbid AD was associated with higher levels of IgE to Asp f 6 (manganese superoxide dismutase from Af, a ubiquitous pan-allergen in fungi) and low but positive levels of IgE to other Af-components, which hampered the serological discrimination of ABPA. CONCLUSIONS AND CLINICAL RELEVANCE: The levels of IgE to Asp f 1 and/or Asp f 2 can effectively differentiate ABPA from Af-sensitized asthma, suggesting that the amounts of IgE specific for these molecules are markers for genuine Af-sensitization in ABPA. However, comorbid AD must be taken into consideration in the interpretation of high IgE to Asp f 6. Establishing of IgE-sensitization profiles using panel of Af-allergen components provides valuable information for distinguishing genuine vs. cross-reactive sensitization in Af-sensitized patients.

Two routes for pollen entering indoors: ventilation and clothes.

BACKGROUND: The route by which pollen enters dwellings has not been clarified. OBJECTIVE: To evaluate the amount of pollen entering dwellings by ventilation and adhesion to textile products. METHODS: The amount of pollen clinging to fabrics (clothes, laundry, and futon bedding) out of doors was measured by quantification of Japanese cedar pollen antigen Cry j 1. The effect of air ventilation on the amount of pollen indoors was also investigated using several neighboring unoccupied apartments with an identical layout while controlling the ventilation conditions. RESULTS: The amount of pollen adhering to futons was especially high. More than half of the pollen on futons or laundry remained on the surface, even after being brushed off by hand or shaken off. Vacuuming laundry and futons after airing out would be an effective way to decrease the amount of indoor pollen. A large amount of pollen entered dwellings through air ducts when the windows were closed and the ventilation fans working. Since most pollen that entered by ventilation remained near the windows, cleaning carefully and frequently near windows could reduce the amount of pollen indoors. CONCLUSIONS: To decrease the amount of pollen indoors, special attention must be paid to textile products and ventilation systems during the pollen season.

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.

Histamine release with a solid-phase-coupled allergen.

Mite allergen was coupled to Sepharose, paper disk or microcrystalline cellulose, and the immobilized allergen was used for histamine release assay in human leukocytes. In terms of the spontaneous release and the reproducibility, the histamine release assay using immobilized allergen was comparable to that using soluble allergen. Histamine release increased progressively with the concentration of the immobilized allergen up to maximal release of histamine which persisted with further increase in immobilized allergen. Histamine release with soluble allergen decreased at higher concentrations of the allergen. The concentration of immobilized allergen required for maximal histamine release was the same as that with soluble allergen, but the maximal release with immobilized allergen was always about 20% lower than that with soluble allergen. Histamine release was not dependent on the density of allergen molecules on Sepharose beads. Although there was a significant correlation between histamine release obtained with a commercial disk, with a disk prepared in our laboratory, and with soluble allergen, the magnitude of the release by both assays using a paper disk was reduced significantly.

Increased activity of 5-lipoxygenase in polymorphonuclear leukocytes from asthmatic patients.

The formation of 5-lipoxygenase products of arachidonic acid, 5-HETE and 5,12-diHETE, was determined in 100,000 X g supernatant of polymorphonuclear leukocytes from 17 healthy subjects, 17 patients with extrinsic asthma and 15 patients with intrinsic asthma. After the supernatant was incubated with 14C-arachidonic acid in the presence of calcium and indomethacin, the lipoxygenase products of arachidonic acid were separated by thin layer chromatography. The results were expressed as the percentage conversion of 14C-arachidonic acid into the product per 10(7) cells. The formation of 5,12-diHETE, but not of 5-HETE, was significantly increased in the cells from the group of patients with extrinsic asthma (4.38 +/- 0.78%, mean +/- S.E.; p less than 0.01) and intrinsic asthma (6.09 +/- 1.11%; p less than 0.01), when compared to normal subjects (1.74 +/- 0.30%). Both extrinsic and intrinsic asthmatics had significantly enhanced 5-lipoxygenase activity, which was expressed as the sum of percentage conversion of 14C-arachidonic acid into 5-HETE and 5,12-diHETE. The percentage conversion in normal subjects was 4.19 +/- 0.39%, 6.24 +/- 0.84% for 17 patients with extrinsic asthma (p less than 0.05), and 8.59 +/- 1.29% for 15 patients with intrinsic asthma (p less than 0.01). There was no significant difference between these asthmatic groups. These results indicate that 5-lipoxygenase activity is increased in patients with bronchial asthma.

Enzymatic dispersion of mast cells from human sinus mucosa: characterization of histamine release and its comparison with chopped fragments of the tissue.

Functional characteristics of mast cells in chopped fragments from sinus mucosa, which was dissected from patients with chronic sinusitis, were compared with those from dispersed cells prepared by enzymatic treatment. The results obtained in this study were the following. (1) Both chopped fragments and dispersed cells released histamine in a dose-dependent manner when incubated with anti-IgE. However, higher histamine release was always observed in dispersed cells. (2) Although no differences in the ability to reduce histamine release with salbutamol or forskolin could be observed between chopped fragments and dispersed cells, staurosporin and p-bromophenacyl bromide were more active on dispersed mast cells than chopped fragments. (3) Passive sensitization of dispersed cells with an allergic serum containing IgE to mite could be achieved only after elution of IgE on the cells with lactic acid.

IgE reactivity and cross-reactivity to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens in dogs with atopic dermatitis.

The natural occurrence of Japanese cedar (Cryptomeria japonica) pollinosis has been reported in dogs with atopic dermatitis. However, the reactivity to Japanese cypress (Chamaecyparis obtusa) pollen allergens in these dogs has not been reported. The present study was designed to investigate the reactivity to Japanese cypress pollen allergens in dogs sensitized to Japanese cedar pollen allergens. In 19 dogs with specific IgE to C. japonica pollen allergen, we measured the specific IgE to C. obtusa pollen allergen and examined the reactivity to the allergen by intradermal test. Of the 19 dogs, 18 had specific IgE to crude and purified major allergens (Cha o 1) of C. obtusa pollen. Most of the dogs showed a positive reaction to C. obtusa pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in C. japonica pollen) was observed by the ELISA inhibition method. Dogs sensitized to Japanese cedar pollen allergens demonstrate reactivity to Japanese cypress pollen allergens.

Analysis of the canine IgE-binding epitope on the major allergen (Cry j 1) of Japanese cedar pollen with anti-Cry j 1 monoclonal antibodies.

In our previous study [Immunology 91 (1997) 161] using monoclonal antibodies (mAbs) specific to Cry j 1, a major allergen in Japanese cedar (Cryptomeria japonica) pollen, we identified five independent epitopes (EP-1-EP-5) on the molecule and found that EP-1 and EP-5 are the predominant allergic epitopes for humans and monkeys, respectively. In this study, we analyzed the epitopes recognized by IgE in the sera of 10 dogs sensitive to C. japonica pollen allergen using an IgE-ELISA inhibition method with these mAbs. The IgE reaction patterns varied among dogs. In eight of the 10 dogs, IgE recognized EP-5 which is a predominant allergic epitope for monkeys with the pollenosis. In four dogs, IgE recognized EP-1 which is a predominant allergic epitope for human patients with the pollenosis. In three dogs, IgE recognized EP-4 which is a heat-stable epitope. EP-5 is a predominant allergic epitope for dogs and some, but not all, dogs have IgE reaction patterns to the epitopes similar to those of humans.

Identification of peptides containing T-cell epitopes of Japanese cedar (Cryptomeria japonica) pollen allergen (Cry j 1) in dogs.

Japanese cedar (Cryptomeria japonica, CJ) pollen has been known to cause atopic dermatitis in dogs in Japan. However, since the mechanism of the CJ antigen recognition is not well understood in dogs, it is difficult to develop effective immunotherapy for atopic dermatitis caused by sensitization to CJ pollen. In order to aim at development of a peptide immunotherapy, we tried to identify T-cell epitopes of a major allergen of CJ pollen, Cry j 1, in dogs sensitive to CJ pollen allergen. Peripheral blood mononuclear cells (PBMCs) obtained from 22 dogs experimentally sensitized to CJ pollen allergen and 5 atopic dogs sensitive to CJ pollen allergen were used for mapping of T-cell epitopes of Cry j 1 using 35 kinds of synthesized overlapping peptides of Cry j 1. Reactive peptides were identified based on the results of blastogenic responses of PBMCs against the peptides when the stimulation indices were beyond 2.0. Three reactive peptides were identical in a relatively high population of experimental dogs, which were Nos. 8 (p71-90) (41%), 10 (p91-110) (50%), and 11 (p101-120) (41%). It was considered that these synthesized peptides should contain T-cell epitopes of Cry j 1 in the dogs. However, there were no reactive peptides identical among the five atopic dogs spontaneously sensitive to CJ pollen. The population of dogs experimentally sensitized to CJ pollen antigen will be used in order to investigate effects of a peptide immunotherapy using the reactive peptides. The results in atopic dogs sensitive to CJ pollen antigen will also provide useful information on necessity to develop a tailor-made immunotherapy using reactive peptides in each dog.

IgE-reactivity to major Japanese cedar (Cryptomeria japonica) pollen allergens (Cry j 1 and Cry j 2) by ELISA in dogs with atopic dermatitis.

The present study investigated IgE-reactivity to two major Japanese cedar (Cryptomeria japonica, C. japonica) pollen allergens (Cry j 1 and Cry j 2) in dogs with atopic dermatitis by use of a fluorometric ELISA. The serum samples from 27 dogs that showed IgE-sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to the two major allergens. All 27 dogs had anti-Cry j 1 IgE, and 10 (37%) had anti-Cry j 2 IgE. Inhibition of binding of dog specific IgE to crude C. japonica pollen allergen was carried out by addition of Cry j 1. When serum samples containing anti-Cry j 1 IgE but no anti-Cry j 2 IgE were incubated with Cry j 1, specific IgE binding to crude C. japonica pollen allergen was almost abolished. These findings suggest that Cry j 1 is a major allergen in dogs.

IgE sensitivity and cross-reactivity to crude and purified mite allergens (Der f 1, Der f 2, Der p 1, Der p 2) in atopic dogs sensitive to Dermatophagoides mite allergens.

The present study investigates IgE-reactivity to crude and purified mite allergens by intradermal skin test (IDST), Immunodot method, and ELISA in atopic dogs sensitive to mite allergens, as well as the allergenic cross-reactivity between Dermatophgoides (D) farinae (DF) and D. pteronyssinus (DP) in dogs by IgE-ELISA inhibition. IDST and Immunodot method for crude mite allergens were performed for atopic dogs and 16 atopic dogs showed sensitivity to mite allergens. Of the 16 dogs, all dogs had anti-DF IgE and 11 had anti-DP IgE. We measured specific IgE to purified major allergens (Der f 1, Der f 2, Der p 1, Der p 2). Of the 16 atopic dogs, six had anti-Der f 1 IgE and seven had anti-Der f 2 IgE. Similarly, of the 16 dogs, six had anti-Der p 1 IgE and seven had anti-Der p 2 IgE. However, eight dogs had no specific IgE to these mite allergens. These dogs may be sensitive to other major mite allergens except Der 1 and Der 2. In the dogs that had both anti-DF and DP IgE, IgE binding to DF was greatly inhibited by DP, and reciprocal inhibition was observed. Based on these data, it appears that there is a strong cross-reactivity between DF and DP in dogs. Similarly, a cross-reactivity between DF and DP in purified allergens was also observed. IDST and Immunodot method are useful methods for the diagnosis of atopic diseases in dogs, and ELISA is a useful method for further investigation of IgE-reactivity for the allergens.

Removal of cat major allergen (Fel d I) from futon (Japanese bedding) with a home washing machine.

We evaluated the removal of a cat major allergen (Fel d I) from futons (Japanese bedding) with the use of a large-sized home washing machine. Before and after washing a futon that had been used in a home with a cat, a small amount of cotton was collected from the futon and Fel d I was extracted from the cotton. The levels of Fel d I were assayed by a sandwich enzyme-linked immunosorbent assay (ELISA). We found that washing reduced the Fel d I level in futons by more than 95%. In conclusion, washing of futons is an effective method for elimination of their cat allergens.

[Special cloth futon-cover (Miroguard) as a protection against house dust mite exposure].

We studied the efficacy of a special cloth encasing (Microguard) in protecting against house dust mite exposure. We vaccumed dust from right or left half surface of a shiki-futon (Japanese style mattress). Then we encased the shiki-futon by a new special encasing, vaccumed dust from the other part of the shiki-futon and got a pair of dust samples. We had done the same of the same shiki-futon by a used special encasing (used for one and half years) for about 2 weeks later. We prepared 7 shiki-futons and collected 14 pair dust samples. We weighed the dust and measured the mite allergens with a monoclonal antibody to Dermatophagoides pteronissynius and Dermatophagoides farinae. The dust level was 1.0% of the control (no encasing) in the new encasing group and 2.0% of the control in the used encasing group. The Der I concentration was 2.5 micrograms/(g dust) in the new encasing group and 3.2 micrograms/(g dust) in the used encasing group. The Der II concentration was 1.6 micrograms/(g dust). The total amount of Der I was 0.1% of the control in the new encasing group and 0.5% of the control in the used encasing group. The total amount of Der II was 0.2% of the control in the new encasing group and 0.7% of the control in the used encasing group. We compared Der p and Der f levels in the dust samples which we assayed and found no significant differences either in Der I or in Der II allergen. We concluded that Microguard was a useful tool to avoid mite allergen exposure by reducing not only the concentration of mite allergens but also the amount of dust.

[Specific IgE antibody titers to hen's egg white lysozyme in allergic children to egg].

Hen's egg white lysozyme (HEL) is one of the minor allergen in hen's egg white. HEL is commonly used to treat disease of respiratory tract, because it have the effect to dissolve mucopolysaccharide and anti-inflammatory action. We examined specific IgE antibody titers (IgE-HEL) in patients with egg allergy and allergic patients to other antigen than egg. Results indicated that 16.37 +/- 29.56 (PRU/ml) (mean +/- SD) of IgE-HEL was found in 30 out of the 39 allergic patients to egg, and 23 (66.7%) out of the 39 patients studied showed RAST scores of more than 2. On the other hand, 1.08 +/- 0.92 (PRU/ml) of IgE-HEL in 12 out of the 44 allergic patients to other antigen than egg, and 5 (11.4%) out of the 44 patients studied showed RAST scores of more than 2. Moreover, we treated a patient who developed anaphylaxis after taking HEL. 1.0 (PRU/ml) of HEL-IgE was found in this patient. These results suggest that we should be careful in treating allergic patients with HEL.

[An enzyme-linked immunosorbent assay (ELISA) for the quantitation of sugi pollen and Dermatophagoides mite allergens and its application for standardization of allergen extracts].

A simple enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of the major allergens of sugi pollen, Cry j I and of Dermatophagoides mites, Der I (Der p I/Der f I) and Der II (Der p II/Der f II) for use in the in vitro standardization of allergen extracts. Polystyrene microplates coated with a IgG fraction of rabbit antiserum were incubated first with allergen extracts and then with biotinylated antiserum IgG. The bound allergen-biotinylated antibody complex was detected with commercially available streptavidin-enzyme conjugate followed by the addition of colorimetric substrate. The assay was very sensitive (-0.2 ng/ml) and reproducible (CV% = 1.9-13.8%). The ELISA was compared with the radioimmunoassay previously described, and the results showed a very good correlation between the assays (r = 0.967-0.990). The allergen content in three sugi pollen and three house dust extracts measured by the ELISA also demonstrated a good agreement with the relative potency of these extracts as determined by the intradermal skin test. These results indicate that the ELISA could be useful in the standardization of allergen extracts.

[Kinetics of the falling of airborne mite allergens (Der I and Der II)].

A futon (Japanese quilt) was beaten to disperse mite allergens into the air in a closed room, and the airborne allergens were collected both by Andersen air sampler for particle size analysis and by slit air sampler for kinetic analysis of the clearing of the allergens from the air. After extraction of the allergens from the agar plates in the samplers, two kinds of major mite allergens (Der I and Der II) were immunochemically quantitated. We found that the aerodynamic diameters of both allergens were mainly above 5.5 microns, and that airborne allergen levels decreased to about 10% of the starting level in 30 minutes, indicating the rapidity of the falling of both allergens.

[Evaluation of different treatments of Japanese bed quilts for reducing mite allergens].

We evaluated the effectiveness of different treatments of Japanese bedquilt (futon) in reducing mite allergens: vacuum-cleaning, beating plus vacuum-cleaning and washing of the whole futon in water. Before and after these treatments, small amounts of cotton were taken out of the futon and mite allergens were extracted from the cotton into water. The absolute contents of two kinds of major allergens of two Dermatophagoides species were immunochemically quantitated. We found that beating and vacuum-cleaning reduced the allergen contents by only about 40%, whereas washing reduced the allergens by more than 90%. Therefore, for reducing airborne mite allergens generated from futon, we think that washing the whole futon in water is the most effective method.

[The diagnostic value of a new whole blood histamine release test using paper disc-coupled antigens--results compared with intracutaneous test, RAST, and eye test].

We have developed a novel histamine release test (HR) using whole blood and antigen-coupled RAST paper discs, for the screening of allergens rather in a short time with a small amount of blood. Histamine was determined by a RIA kit. In order to evaluate the diagnostic value, the results of the test were compared with those of RAST, intracutaneous tests, and eye tests in 45 allergic patients. HR correlates better with RAST than intracutaneous test in almost all antigens. The closest positive correlation with the other tests was seen in mite allergen, followed by pollen, foods and mould spores in that order. When the HR was positive for house dust, 100% of the patients were also positive for the eye test, which is reported to closely correlate with bronchial provocation tests. HR seemed to be a useful test not only for the screening but also for the determination of pathogenic allergens.

[Changes in antigen-specific IgG, IgG4, and IgE antibodies in patients receiving immunotherapy with house-dust extract].

We measured allergen-specific IgG, IgG4, IgE antibodies and total IgE in sera from 64 patients who were receiving conventional immunotherapy (IT) with house dust (HD) extract. The sera were taken before and after IT. To measure allergen-specific antibodies, we used crude DF antigen, Der f I and Der f II for IgG and IgG4 antibodies, and crude DF antigen for IgE antibodies. The patients were divided into 3 groups: very short term group (IT period < 2 years, n = 9); short term group (2 < = IT period < 7 years, n = 25); long term group (7 years < = IT period, n = 30). The specific IgG antibodies for DF crude antigen did not change in any group. The specific IgG antibodies for Der f I and Der f II increased in the short term group and in the long term group. The specific IgG4 antibodies for all three antigens increased remarkably in the short term group and in the long term group. In the very short term group, however, the specific IgG4 antibodies did not increase for any antigen. Total IgE and the specific IgG antibodies did not change through IT. The specific IgG4 antibodies increased significantly through IT, but the time course of the increment of the IgG4 antibodies was not parallel with the clinical course of IT.