Analysis of sequence polymorphism of a major mite allergen, Der p 2

Background The major house dust mite allergen Der p 2 has been regarded as an important allergen involved in the immunopathogenesis of allergic asthma and eczema. Objectives To determine the degree of sequence polymorphism exists in Der p 2 at both the genomic DNA and cDNA levels. Methods Isolation of cDNA clones was performed by screening the cDNA libraries with Der p 2 cDNA probe and the genomic sequences for Der p 2 were obtained by PCR amplification from environmental dust mites with Der p 2-specific primers. DNA sequencing was carried out by the dideoxynucleotide chain termination method. The study of Der p 2 isoforms was performed by 2-D gel immunoblot analysis using mouse anti-Der p 2 serum. Results In this study, we have characterized the seqtiences of three difierent genealleles coding for the major house dust mite allergen Der p 2 at the cDNA level. The translated polypeptides from these clones differed from each other by 3–4 amino-acid residues. These polymorphic residues determined were also found in Der f 2 and they were located in regions containing T-epitopes. In addition, the genomic DNA sequences of Der p 2 which were obtained by PCR amplification using the environmental mites isolated from Taiwan and Australia have helped to confirm the authenticity of the polymorphisms detected in the cDNA clones generated from CSL cultured mites. Furthermore, 2D- immunoblot analysis indicated that there were at least 10 different isoforms (p 1 values range from greater than 7.0–5.9) of Der p 2 proteins produced by CSL cultured mites. Conclusion The results showed that there was a small but significant degree of sequence polymorphisms exists in Der p 2 gene alleles. Interestingly, the polymorphic residues were found in regions containing previously determined T-epitopes. The polymorphism data reported here will be important for the understanding of the immune responses of mite allergens as well as for the development of the peptide-based immunotherapeutic reagents for mite allergy.     

Reduction in allergenicity of grass pollen by genetic engineering

BACKGROUND: Hay fever and allergic asthma triggered by grass pollen allergens affect approximately 20% of the population in cool temperate climates. Ryegrass is the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grass pollen allergen because it reacts with IgE antibodies of more than 90% of grass pollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made in developing diagnostic and therapeutic reagents for designing new and more effective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of allergen content in the source plant. METHODS: High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one of major allergen, Lol p 5, was introduced. The expression of antisense RNA was regulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity. RESULTS: Analysis of proteins with allergen-specific monoclonal and polyclonal antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE binding capacity of pollen extract as compared to control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development. CONCLUSIONS: Our studies showed that it is possible to selectively 'switch off' allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity.     

Proteolytic activity of the house dust mite allergen Der p 1 enhances allergenicity in a mouse inhalation model

BACKGROUND: We have recently demonstrated that intraperitoneal immunization of mice with proteolytically active Der p 1, the major house dust mite allergen, results in a significant and selective enhancement of total and Der p 1-specific IgE synthesis compared to mice immunized with proteolytically inactive Der p 1. OBJECTIVE: To investigate whether the proteolytic activity of Der p 1 would lead to enhanced inflammatory cellular infiltration of the lungs and systemic IgE production when administered through the respiratory system, which is the natural route of entry for this allergen. METHODS: Groups of mice were initially sensitized with proteolytically active Der p 1 through the intraperitoneal and the subcutaneous routes and subsequently exposed intranasally to either proteolytically active Der p 1, inactive Der p 1 or PBS. The extent of cellular infiltration of the lungs and systemic IgE production in the three animal groups were then compared. RESULTS: Here, we show for the first time that the administration of proteolytically active Der p 1 to mice through the intranasal route leads to significant inflammatory cellular infiltration of the lungs and systemic production of IgE. CONCLUSIONS: These data underline the important role of the proteolytic activity of Der p 1 in driving the allergic response in the lungs.     

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.     

IgE binding structures of the major house dust mite allergen Der p I.

The group I allergens Der p I and Der f I are potent allergens of mites from the genus Dermatophagoides. IgE radioimmune dot blots and immunoabsorption with recombinant peptides have been used to define areas of antigenicity. Four linear binding regions comprising residues 15-33, 60-80, 81-94 and 101-111 were found in the N terminal domain and one, 155-187, in the C-terminal domain, but direct evidence for their discontinuous nature is shown. Firstly, the binding activity of residues 60-80 required either C- or N-terminal flanking sequences to express reactivity and secondly a discontinuous determinant was directly demonstrated by the two non-overlapping peptides 53-99 and 101-154 which significantly cross absorbed specificities to one another. This also indicates considerable homogeneity in the antibodies recognising these peptides. The IgE binding peptides could be located to equivalent residues on the X-ray crystallographic structure of the homologous proteins actinidin and papain. The residues 81-94 and 101-111 which gave strong reactivity were located on a flexible loop connecting the domains and represent areas in which synthetic peptides could be expected to retain activity.     

Decay of house-dust mite allergen Der f 1 at indoor climatic conditions.

BACKGROUND: The decay of house-dust mite allergens is important for the outcome of avoidance measures for house-dust mite-allergic patients. OBJECTIVE: To quantify the stability of Der f 1 from mattress dust when exposed to domestic conditions. METHODS: Three samples of mattress dust were individually homogenized and divided into 64 subsamples. Mites were killed by freezing for 48 hours at -30 degrees C. The subsamples were exposed in eight homes, three storerooms, and one greenhouse, where temperature and relative humidity were recorded. Der f 1 was determined in extracts of subsamples (enzyme-linked immunoadsorbent assay) at 0, 3, 12, and 24 months. RESULTS: In the three samples of mattress dust, the initial concentrations of Der f 1 (mean +/- standard deviation; STD) were: 169 (12), 3.9 (0.4), and 31 (2.6) microg/g, respectively. Median half-life of Der f 1 in the mattress dust samples was 10 years in the exposure homes, 18 years in the store rooms, and 1.0 year in the greenhouse. No correlations among preserved Der f 1 and temperature, relative humidity, and absolute humidity in homes were found (Spearman rank correlation test). CONCLUSION: Natural decay of Der f 1, with an estimated half-life of 10 years at housing conditions, has no practical consequence in reducing allergen exposure. Therefore, avoidance measures should include an active removal of the allergens.     

cDNA encoding the major mite allergen Der f II

cDNA encoding the major house dust mite allergen Der f II from Dermatophagoides farinae was amplified using the polymerase chain reaction and cloned into E. coli. It encoded a 129-residue protein with a calculated molecular weight of 14,021 D and had the expected high homology (88%) with Der p II including the absence of N-glycosylation sites and conserved cysteine residues. These results are consistent with the high degree of antibody crossreactivity and may help identify the differences in T-cell epitopes revealed for these molecules so far.     

Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen,by human dendritic cells

BACKGROUND: Immature dendritic cells (DCs) take up antigens in peripheral tissues and, after antigen processing, mature to efficiently stimulate T cells in secondary lymph nodes. In allergic airway diseases DCs have been shown to be involved in the induction and maintenance of a T(H)2-type profile. OBJECTIVE: The present study was undertaken to determine pathways of Der p 1 (a house dust mite allergen) uptake by human DCs and to compare Der p 1 uptake between DCs from patients with house dust mite allergy and DCs from healthy donors. METHODS: Monocyte-derived DCs (MD-DCs) were obtained from patients with house dust mite allergy (n = 13) and healthy donors (n = 11). Der p 1 was labeled with rhodamine. Der p 1 uptake by MD-DCs was analyzed by means of flow cytometry and confocal microscopy. RESULTS: Rhodamine- labeled Der p 1 was demonstrated to be taken up by MD-DCs in a dose-, time-, and temperature- dependent manner. The involvement of the mannose receptor (MR) in the Der p 1 uptake was demonstrated by using (1) inhibitors of the MR- mediated endocytosis (mannan and blocking anti-MR mAb), which inhibited the Der p 1 uptake from 40 % to 50 %, and (2) confocal microscopy showing the colocalization of rhodamine-labeled Der p 1 with FITC-dextran. Interestingly, compared with DCs from healthy donors, DCs from allergic patients expressed more MR and were more efficient in Der p 1 uptake. CONCLUSION: These results suggest that the MR could play a key role in the Der p 1 allergen uptake by DCs and in the pathogenesis of allergic diseases in dust mite -sensitive patients.     

Exposure to house-dust mite allergen (Der p I) and the development of asthma in childhood. A prospective study

BACKGROUND AND METHODS. Children with asthma commonly have positive skin tests for inhaled allergens, and in the United Kingdom the majority of older children with asthma are sensitized to the house-dust mite. In a cohort of British children at risk for allergic disease because of family history, we investigated prospectively from 1978 to 1989 the relation between exposure to the house-dust mite allergen (Der p I) and the development of sensitization and asthma. RESULTS. Of the 67 children studied in 1989, 35 were atopic (positive skin tests), and 32 were nonatopic. Of the 17 with active asthma, 16 were atopic (P less than 0.005), all of whom were sensitized to the house-dust mite, as judged by positive skin tests and levels of specific IgE antibodies (P less than 0.001). For house-dust samples collected from the homes of 59 of the children in 1979 and from 65 homes in 1989, the geometric means for the highest Der p I exposure were, respectively, 16.1 and 16.8 micrograms per gram of sieved dust. There was a trend toward an increasing degree of sensitization at the age of 11 with greater exposure at the age of 1 (P = 0.062). All but one of the children with asthma at the age of 11 had been exposed at 1 year of age to more than 10 micrograms of Der p I per gram of dust; for this exposure, the relative risk of asthma was 4.8 (P = 0.05). The age at which the first episode of wheezing occurred was inversely related to the level of exposure at the age of 1 for all children (P = 0.015), but especially for the atopic children (r = -0.66, P = 0.001). CONCLUSIONS. In addition to genetic factors, exposure in early childhood to house-dust mite allergens is an important determinant of the subsequent development of asthma.     

Murine allergic respiratory responses to the major house dust mite allergen Der p 1.

BACKGROUND: Although many studies have examined chronic asthma, limited data exist on acute immunopathogenic events induced by allergens. The aim of the study was to investigate the acute cellular, serologic and histopathologic events in airway inflammation produced by intranasal challenge of mice sensitised to the major house dust mite allergen Der p 1. METHODS: C57BL/6 mice were immunised subcutaneously with Der p 1 in alum. Mice were bled and challenged intranasally with Der p 1 on day 14 and killed on day 17. Lungs were fixed in situ, processed and stained with haematoxylin and eosin. The degree of inflammation and eosinophil infiltration was quantified by image analysis. Specific IgE was determined by passive cutaneous anaphylaxis. Cells from spleen and draining lymph nodes were cultured for 24 h with Der p 1, and IL-3/GM-CSF released into supernatants was measured by bioassay. RESULTS: Intranasal challenge of sensitised mice induced eosinophilic influx into the large and small airways and the alveolar regions of the lung, mucus plugging and in severe cases numerous Charcot-Leyden crystals. The quantitation of the inflammation induced by different sensitisation and challenge doses showed that optimal inflammation could be produced using only 1 microg of allergen for both sensitisation and challenge. The degree of inflammation was not related to the titre of IgE antibody and was indeed produced in its absence. T cell reactivity of spleen cells to the allergen was decreased suggesting cell migration or inactivation. CONCLUSIONS: Mice sensitised and challenged intranasally with as little as 1 microg of Der p 1 produced an extensive pulmonary eosinophilic inflammation which shared many of the features of the inflammation found in asthma. The small amount of allergens required and the use of intranasal challenge should provide a useful model.     

IgE and monoclonal antibody binding by the mite allergen Der p 7

Background The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergie patients. Objective This study was performed to compare the IgE bindinig activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies. Methods IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay. Reslts A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f 7 was used for epitope analysis. All of these reaeted with eaeh allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, lour could inhibit the binding of IgE (WH9, WH22. WPS and HD19) while one (WH14) could not. Conclusions Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specilieities.     

Prediction of mite allergen levels by guanine measurements in house-dust samples

We studied the sensitivity and specificity of guanine environmental tests in the evaluation of the two mite allergen levels, i.e. 2 üg/g and 10 üg/g of Der p I and Der f I, considered to be risk factors for sensitization or for the development of acute asthma. We examined 239 house-dust samples for Der p I and Der f I levels (ELISA) and guanine contents (semiquantitative guanine test and quantitative assays). All house-dust samples with class 2 or 3 guanine tests contained more than 2 üg/g of Der p I and Der f I. The probability that house-dust samples of class 2 contained more than 10 üg/g of mite allergens was 88%; it was 100% for house-dust samples of class 3. The probability that a house-dust sample of class 0 contained less than 2 üg/g of mite allergen was 87%. For each level of mite allergen, a ROC curve was constructed with the true positive rates and the false positive rates calculated by different cutoffs of guanine concentration. The cutoff point which gave the best compromise between sensitivity (76%) and specificity (89%) was 2100 üg/g for the threshold of 2 üg/g of Der p I and Der f I. For detection of a mite allergen > 10 ųg, a guanine content of 3000 üg/g gave the best compromise between sensitivity (86%) and specificity (93%). In conclusion, the guanine test represents a satisfactory environmental test, inexpensive and simple, for predicting mite allergen levels. Semiquantitative and quantitative guanine measurements are more accurate in predicting an exposure level of 10 üg/g than of the level 2 üg/g of Der p I and Der f I.     

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.     

Sequence polymorphisms of the Der p 3 house dust mite allergen

Background The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectricfocusing studies with the natural Der p 3 protein have indicated that several isoforms exist. Objective To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. Methods Five cDNA clones of Der p 3 have been isolated from a λ gt 10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco RI digested genomic DNA was performed. Results Southern blot analysis of Eco RI digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones ditfered only in their 3′untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. Conclusions These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group I and group 2 house dust mite allergens.