Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies

The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity-purified Der p 7-GST (glutathione S-transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5-GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2-D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts.     

Characterization of the allergen Der f 7 from house dust mite extracts by species-specific and crossreactive monoclonal antibodies

Background The group 7 mite allergens react with IgE in 50% of sera from allergic patients. Objective To determine the molecular and antigenic characteristics and heterogeneity of Der f 7 in mite extracts. Methods Monoclonal antibodies (MoAbs) produced from mice immunized with recombinant Der f 7 were examined for crossreactivity to Der p 7 and then used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosylation was studied with N-glycosidase-F and N-terminal sequencing by Edman degradation. The epitopes of the monoclonal antibodies were compared by cross-inhibitory immunoassays. Results Immunoblotting of D. farinae extracts with all the anti Der f 7 MoAbs showed major reactivities at 31, 30 and 25 kDa. The strongest immunostaining was at 25 kDa which contrasted with Der p 7 where the 31 and 30 kDa bands were strongest. The relative strength of staining however varied between extracts. The 31 and 30 kDa components were glycosylation products of the 25 kDa form which had the N-terminal sequence predicted from cDNA analysis. Two MoAbs stained an 18 kDa band consistent with a degradation product. The 2-D gels showed that different components with pls from 5.6–6.4. Both species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed with MoAbs recognizing different epitopes. Conclusions Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently exists as different glycosylation and degradation products in mite extracts, the relative abundance of which differs with different preparations. A two-site ELISA to measure the allergen was developed.     

Cockroach allergen in house dust

The cockroach allergen (Bla g 1) content was determined in the floor dust of 46 homes with recent cockroach extermination in Amsterdam, The Netherlands. IgE antibodies to Blattella germanica , house-dust mite, cat dander, dog dander, and a mixture of molds were determined in venous blood samples of 46 children (4-12 years) and one of their biologic parents (24-54 years). Specific IgE to cockroach was also determined in a sample of the general population studied in a previous case-control study, one group ( n =20) with and three groups ( n =76) without history of cockroach infestation of the home. Cockroach allergen was detected in floor dust from 44% of the homes, with levels up to 3899 ng Bla g 1/g. Seven of the 46 adults and only one of the 46 children studied had positive RAST to cockroach. Geometric mean cockroach allergen concentrations in living room and master bedroom of sensitized adults were similar to those of nonsensitized adults. In the groups of children without a history of cockroach infestation of the home, positive RAST against cockroach was observed in 16% of the children with respiratory symptoms, in 4% of the children without respiratory symptoms, and in 48% of the children with two or more positive RAST to other allergens. Of the 18 children with positive RAST against cockroach, only one had a history of cockroach infestation of the home and 16 (89%) had also positive RAST against house-dust mite.     

Exposure to house dust mite allergen of children admitted to hospital with asthma

Eighty-two children admitted to hospital with exacerbations of asthma were studied to determine how many were exposed to house dust mites at the time of admission and displayed immediate hypersensitivity to house dust mites. The concentration of house dust mite allergen (Der p I) was measured in dust obtained from the child's mattress, bedroom floor and living room floor. Sixty-two (75%) children admitted had been exposed to > 10 microg Der p I/g. Sixty-seven (82%) children were sensitive to house dust mite (RAST > or = 1 +, or weal > or = 3 mm): 49 (60%) children were both exposed and sensitive. In contrast in a control group of 44 children, 31 (70%) (n.s.) were exposed to > 10 microg Der p I/g, 10 (23%) (P<0.001) were sensitive to house dust mite, and 7 (16%) (P<0.001) were both exposed and sensitive. Seventy-three homes were revisited 6 months after the child's initial admission. During the preceding month 14 children had been readmitted, 12 were fully investigated; of these 10 were both sensitive to house dust mite and still exposed to > 10 microg Der p I/g. In contrast, of the remaining 62 children who were not readmitted, only 19 were both sensitive and still exposed to > 10 microg Der p I/g (P<0.001). In conclusion, the majority of children admitted to hospital with exacerbations of asthma were exposed to house dust mite allergen and were house dust mite sensitive. Further the results suggest that continued exposure to higher concentrations of mite allergen may be associated with the risk of readmission.     

Complement activation by house dust: reduced reactivity of serum complement in patients with bronchial asthma.

Among ten different allergens, house-dust extract proved to be the most potent complement activator. It was therefore chosen to investigate the susceptibility of complement in the serum and bronchoalveolar lavage fluid of patients with extrinsic asthma and control persons. Complement activation in serum was assessed by the appearance of C3d as well as the activation-specific protein-protein complexes C1rs-C1inhibitor (classical pathway) and C3b(Bb)P (alternative pathway). Complement was activated via both the classical and the alternative pathway in a dose- and time-dependent manner. In contrast to earlier observations, however, complement was less affected in the serum of asthmatics than in the serum of normal individuals. Differences were restricted to alternative-pathway activation, probably due to preactivation and/or a significantly higher serum concentration of the regulatory protein factor H in asthmatic patients. In vitro generation of C3a in bronchoalveolar lavage fluid could not be achieved, although the presence of alternative pathway proteins C3, B and D was demonstrated.     

Distribution of house dust mite allergen: comparing house dust mite allergen levels in dust samples collected from different sites on living room floors with smooth coverings

BACKGROUND: The distribution of house dust mite allergen (Der p1) in living rooms with smooth floor coverings, as measured in the middle compared with the border of the floor was investigated. It was hypothesized that activity causes displacement of Der p1, from the middle towards the border. METHODS: Dust samples from the middle and border of 50 floors with smooth coverings were collected and analysed on Der p1 content in a standardized way. RESULTS: The Der p1 exposure expressed as per unit area (ng/m2) showed that border samples contained significantly more Der p1 compared with middle samples (median: 2.57 vs 0.27, respectively, P = 0.023). Presence of pets and presence of more than two inhabitants increased the difference. When expressed as per unit weight of dust (ng/g), significant differences were only detected when comparing Der p1 content of samples collected in households with three or more inhabitants [median: 2 (border) vs 53 (middle), respectively; P = 0.035]. CONCLUSIONS: The Der p1 is unequally distributed on living room floors with smooth coverings, most likely because of displacement of dust from the middle towards the border due to activity. Expression as ng/g of dust and ng/m2 could not obviously be interchangeable.     

屋尘螨变应原重组体免疫治疗的实验研究

目的：建立屋尘螨哮喘动物模型，比较分别用屋尘螨粗浸液、重组Derp2（rDerp2）进行免疫治疗的疗效，为变态反应性疾病的免疫治疗提供新的思路。方法：近交系清洁级BALB／c小鼠，腹膜内注射（i．p．）屋尘螨变应原粗浸液与灿（OH）3生理盐水的混悬液致敏小鼠，建立哮喘模型；治疗组在致敏后分别给予i．p．屋尘螨粗浸液、rDerp2溶液进行免疫治疗后，鼻腔滴入（i．n．）屋尘螨粗浸液予以激发；阴性对照组从致敏，阳性对照组在致敏后，均同步生理盐水处理。用ELISA方法检测各组实验动物血清中IgE、IgG1、IgG2a的水平，以及支气管肺泡灌洗液（BALF）与脾细胞培养后上清液（SCCS）中细胞因子IL-4、IFN-γ水平，计数BALF中细胞总数与分类细胞数，并制备肺组织病理切片以观察其病理改变。结果：添加铝佐剂的屋尘螨变应原致敏的小鼠在特异性变应原的连续激发下，肺组织产生明显的炎症反应，表现为BALF中细胞总数增加以及炎症细胞所占比例及绝对计数升高，且以分泌Th2型细胞因子如IL4为主；血清IgE、IgG1水平升高；肺组织病理切片观察到支气管、细支气管上皮下和小血管周围有明显的炎性细胞浸润，以嗜酸性粒细胞为主，气道上皮结构紊乱，有部分脱落，上皮下充血水肿明显，肺泡间质增厚，也可见到炎性细胞浸润。给予相应免疫治疗后，肺组织炎症反应明显减轻，表现为BALF中细胞总数减少，炎症细胞数减少，且其和SCCS中n1型细胞因子如IFN-γ占主导；血清IsE、IsG1水平降低，IgG2a水平升高；肺组织病理切片观察到细胞浸润程度明显减轻，气道结果基本恢复正常。结论：本实验研究成功地建立了小鼠哮喘模型。分别用屋尘螨粗浸液及rDerp2进行免疫治疗均显示良好疗效，但前者比后者疗效好。     

The allergen of Ficus benjamina in house dust

Ficus benjamina, a member of the Moraceae family, is a tropical, non-flowering green plant which is widely used for ornamental purposes. It is an occupational allergen in plant keepers but sensitization is also increasingly found in non-occupationally exposed atopic and non-atopic patients. The allergen of Ficus benjamina is located in the plant sap, also-called latex. By radioallergosorbent test-(RAST)-inhibition studies allergen could also be demonstrated in the dust collected from the leaf surface and in dust samples from the floor of rooms where the plant was placed. These findings could result in more extensive preventive measures in patients sensitized to Ficus benjamina, In addition there is some evidence that possibly a crossreactivity between latex of Ficus benjamina and latex from the rubber tree Hevea brasiliensis, a member of the Euphorbiaceae family, may exist.     

Effect of improved home ventilation on asthma control and house dust mite allergen levels

Background:  The warm, humid environment in modern homes favours the dust mite population, but the effect of improved home ventilation on asthma control has not been established. We tested the hypothesis that a domestic mechanical heat recovery ventilation system (MHRV), in addition to allergen avoidance measures, can improve asthma control by attenuating re-colonization rates.
Methods:  We conducted a randomized double-blind placebo-controlled parallel group trial of the installation of MHRV activated in half the homes of 120 adults with asthma, allergic to Dermatophagoides pteronyssinus . All homes had carpets steam cleaned and new bedding and mattress covers at baseline. The primary outcome was morning peak expiratory flow (PEF) at 12 months.
Results:  At 12 months, the primary end-point; change in mean morning PEF as compared with baseline, did not differ between the MHRV group and the control group (mean difference 13.5 l/min, 95% CI: −2.6 to 29.8, P  = 0.10). However, a secondary end-point; evening mean PEF, was significantly improved in the MHRV group (mean difference 24.5 l/min, 95% CI: 8.9–40.1, P  = 0.002). Indoor relative humidity was reduced in MHRV homes, but there was no difference between the groups in Der p 1 levels, compared with baseline.
Conclusions:  The addition of MHRV to house dust mite eradication strategies did not achieve a reduction in mite allergen levels, but did improve evening PEF.     

The effect of changes in house dust mite allergen exposure on the severity of asthma

To measure the association between changes in house dust mite (HDM) allergen Der p I exposure and changes in the severity of asthma, we re-analysed data from a clinical trial in which 34 HDM-allergic subjects with asthma (18 women, mean age 35 years) were followed for between 3 and 12 months. The concentration of Der p I in fine dust from the bed, the bedroom floor and the living room floor was measured at 3-monthly intervals along with assessment of subjects' spirometric function and airway hyperresponsiveness (AHR, measured by histamine inhalation test). Daily symptom scores, morning peak expiratory flow rate and peak flow variability were measured throughout the study period. The mean Der p I concentration in the bed at baseline was 25.4 μg/g (95% CI: 15.8–40.6). During the course of the study large within-subject fluctuations were observed in allergen concentrations and in the measurements of the severity of asthma. Changes in allergen concentration in the bed were significantly correlated with changes in AHR (P= 0.003) and symptom score (P= 0.04). Changes in allergen concentration in the living room floor were correlated with changes in symptom scores (P= 0.01). Although these correlations were significant the magnitude of the effect was relatively modest. We conclude that a large reduction in HDM allergen concentration, particularly in the bed, results in a modest reduction in AHR and improvement in symptoms in HDM-allergic subjects with asthma.     

Inhalation provocation test with house dust allergen using tidal breathing

Using the modified 3-Hz oscillation method, the inhalation challenge test was performed with house dust allergen upon subjects while tidal breathing throughout the test. Their total respiratory resistance (Rrs) was continuously monitored with the inhalation of saline as a control, and 250-, 50-, and 10-fold diluted allergen solution to the inhalation of aerosolized metaproterenol. The starting point of the induced bronchoconstriction was checked and as soon as Rrs increased up to twice the base-line value, the subjects inhaled the aerosolized metaproterenol. From the dose-response curve of Rrs, the dose of inhaled allergen (minimum dose) as bronchial sensitivity was determined; also the decreasing rate of respiratory conductance, which was calculated from the slope of elevation of Rrs, as bronchial reactivity. It was found that the more diluted allergen required for a positive skin reaction, the more likely the patients were to have a subsequent positive bronchial inhalation challenge to house dust allergen. There was a relationship between the increased bronchial reactivity and the increased RAST score. These results indicate that this inhalation challenge test is specific, safe, and time-saving.     

Lymphocyte activation by phytohaemagglutinin and pokeweed mitogen. Identification of proliferating cells by monoclonal antibodies

Using a two-colour immunofluorescence technique, we have investigated the mitogenic effects of phytohaemagglutinin-M (PHA-M) and of pokeweed nitrogen (PWM) on human lymphocyte subsets. These were identified by CD1, CD3, CD4, CD8, and CD16 monoclonal antibodies, and proliferation was demonstrated by a polyclonal anti-transferrin antibody. Evidence has been obtained for the generation of a population expressing both the CD4 and CD8 antigens simultaneously, in short-term cultures of peripheral blood mononuclear cells in the presence of PWM and of PHA-M.     

Natural exposure and serum antibodies to house dust mite of mite-allergic children with asthma in Atlanta

Pyroglyphid mites in house dust are important allergens associated with asthma in Europe, but comparable studies of house dust mites in the homes of patients with asthma have not been done in the United States. We examined the distribution of mites and mite allergen in the houses of 20 mite-sensitive children with asthma in Atlanta and measured IgE antibodies to mite allergens in their sera. One or more dust samples from bedding, bedroom floor, television room floor, or television room furniture from 17/20 houses contained greater than 10,000 ng of antigen P1 equivalent per gram of fine dust; amounts ranged from 280 to 230,400 ng/gm. Allergen levels were higher in dust samples from furniture and bedding than from floors. Dust samples obtained from houses in June to September had more mites and mite allergen than those houses sampled in March to April; relative humidity in the room also was higher in June to September. Mite numbers and allergen in floor and furniture samples were correlated with relative humidity in the room and were high when relative humidity was greater than 50%; antigen P1 equivalent was greater than 10,000 ng/gm in 21/39 such samples. Dermatophagoides pteronyssinus was present in all houses and dominant in 11/20. D. farinae was found in 17 houses and was dominant in six. All children studied had high IgE antibody with either D. farinae or D. pteronyssinus RAST; 16 of the 20 children also had IgE to antigen P1. It is likely that the IgE antibody responses in these 20 children with asthma were a direct result of exposure to high levels of mite allergen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cat allergen content of commercial house dust extracts: Comparison with dust extracts from cat-containing environment

Eighteen lots of house dust extract from nine commercial sources (obtained as weight per volume or protein nitrogen unit per cubic centimeter) were analyzed for cat allergen content by direct quantitative immunoelectrophoresis after concentration. Cat allergen 1 was measurable (greater than 0.3 units) in 11 extracts with a mean (range) of 5.8 (1.3 to 31.0) U/gm of source material. Cat albumin was measurable (greater than 2.4 units) in 12 extracts with a mean (range) of 53.4 (11.5 to 319.7) U/gm. In order to evaluate whether the cat allergen 1 content is a significant contribution to the allergenic activity of the extract, 17 cat-allergic subjects were tested by prick test with a purified preparation of cat allergen 1. The mean (range) concentration that produced a 3 mm wheal was 0.01 (0.0013 to 1.33) U/ml. Therefore, the commercial house dust extracts studied, when these extracts were diluted to a concentration commonly used for prick testing, would frequently contain enough cat allergen 1 to produce strong prick test reactions in cat-allergic subjects. It is difficult to justify the use of such commercial dust extracts as diagnostic reagents. For comparison purposes, nine dust samples from an apartment housing two cats were similarly analyzed. Cat allergen 1 was measurable in seven samples with a mean (range) of 23.8 (1.8 to 64.3) U/gm. Cat albumin could be measured in all nine samples with a mean (range) of 32.3 (0.16 to 70.8) U/gm. The average amount of cat allergen 1 that could be washed off the surface of the cats was 270 units. Large reservoirs of cat allergen 1 were present.(ABSTRACT TRUNCATED AT 250 WORDS)     

An experimental model of allergic asthma in cats sensitized to house dust mite or bermuda grass allergen

BACKGROUND: Animal models are used to mimic human asthma, however, not all models replicate the major characteristics of the human disease. Spontaneous development of asthma with hallmark features similar to humans has been documented to occur with relative frequency in only one animal species, the cat. We hypothesized that we could develop an experimental model of feline asthma using clinically relevant aeroallergens identified from cases of naturally developing feline asthma, and characterize immunologic, physiologic, and pathologic changes over 1 year. METHODS: House dust mite (HDMA) and Bermuda grass (BGA) allergen were selected by screening 10 privately owned pet cats with spontaneous asthma using a serum allergen-specific IgE ELISA. Parenteral sensitization and aerosol challenges were used to replicate the naturally developing disease in research cats. The asthmatic phenotype was characterized using intradermal skin testing, serum allergen-specific IgE ELISA, serum and bronchoalveolar lavage fluid (BALF) IgG and IgA ELISAs, airway hyperresponsiveness testing, BALF cytology, cytokine profiles using TaqMan PCR, and histopathologic evaluation. RESULTS: Sensitization with HDMA or BGA in cats led to allergen-specific IgE production, allergen-specific serum and BALF IgG and IgA production, airway hyperreactivity, airway eosinophilia, an acute T helper 2 cytokine profile in peripheral blood mononuclear cells and BALF cells, and histologic evidence of airway remodeling. CONCLUSIONS: Using clinically relevant aeroallergens to sensitize and challenge the cat provides an additional animal model to study the immunopathophysiologic mechanisms of allergic asthma. Chronic exposure to allergen in the cat leads to a variety of immunologic, physiologic, and pathologic changes that mimic the features seen in human asthma.