An in vitro model of peptide-mediated immunomodulation of the human T cell response to Dermatophagoides spp (house dust mite)

Allergic sensitivity of Dermatophagoides spp (house dust mites) is mediated by specific IgE antibody, the production of which requires the presence of CD4+ helper T cells. Attempts to hyposensitize this response in allergic individuals have depended on the administration of extracts of specific allergen. However, the ability of peptides derived from unrelated antigens to inhibit specific immune responses offers an alternative approach to therapy. We have addressed this question by examining the ability of a nonstimulatory peptide analogue derived from influenza virus hemagglutinin to modulate T cell recognition of house dust mite. The peptide inhibited the response of mite-specific CD4+ T cell clones restricted by either the HLA-DRAB1 or DRAB3 gene products. Furthermore, mite-induced polyclonal T cell responses were negatively modulated by the peptide, whereas recognition of common recall antigens remained intact. The inhibitory effects were mediated at the level of the antigen-presenting cell, since no inhibition of mitogen or anti-CD3 antibody-driven T cell proliferation was observed. In direct binding assays, the peptide analogue bound to selected HLA-DR molecules expressed on the membrane of antigen-presenting cells, with specificity predominantly for those class II proteins capable of restricting house dust mite-allergen T cell recognition.     

HLA-DPB1 alleles in house dust mite allergic patients.

Associations were sought between the presence of allergic sensitivity to Der p 1, a major allergen from the house dust mite, and the HLA-DPB1 genotype. Whilst allergic patients did not differ from controls in DPB1 allelic distribution, there was a correlation of DPB1*11011 with strongly reactive T-cell proliferative responses to Der p 1 and high titre specific IgE to Dermatophagoides pteronyssinus.     

An in vitro model of peptide-mediated immunomodulation of the human T cell response to Dermatophagoides spp (house dust mite)

Allergic sensitivity of Dermatophagoides spp (house dust mites) is mediated by specific IgE antibody, the production of which requires the presence of CD4⁺ helper T cells. Attempts to hyposensitize this response in allergic individuals have depended on the administration of extracts of specific allergen. However, the ability of peptides derived from unrelated antigens to inhibit specific immune responses offers an alternative approach to therapy. We have addressed this question by examining the ability of a nonstimulatory peptide analogue derived from influenza virus hemagglutinin to modulate T cell recognition of house dust mite. The peptide inhibited the response of mite-specific CD4⁺ T cell clones restricted by either the HLA-DRABI or DRAB3 gene products. Furthermore, mite-induced polyclonal T cell responses were negatively modulated by the peptide, whereas recognition of common recall antigens remained intact. The inhibitory effects were mediated at the level of the antigen-presenting cell, since no inhibition of mitogen or anti-CD3 antibody-driven T cell proliferation was observed. In direct binding assays, the peptide analogue bound to selected HLA-DR molecules expressed on the membrane of antigen-presenting cells, with specificity predominantly for those class II proteins capable of restricting house dust mite-allergen T cell recognition.     

HLA-DPB1 alleles in house dust mite allergic patients

Associations were sought between the presence of allergic sensitivity to Der p 1, a major allergen from the house dust mite, and the HLA-DPB1 genotype. Whilst allergic patients did not differ from controls in DPB1 allelic distribution, there was a correlation of DPB1*11011 with strongly reactive T-cell proliferative responses to Der p 1 and high titre specific IgE to Dermatophagoides pteronyssinus.     

Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus

T-cell epitopes of Der p II, a major allergen of Dermatophagoides pteronyssinus , were analyzed by using human T-cell clones. We tested 38 cloned T cells from two Japanese patients with allergic rhinitis, and identified at least two peptides (K33-T47 and 158-C73) as helper T-cell epitopes. The former epitope was shown to be restricted by HLA-DRB1* 1502, and the latter by HLA-DRB1* 0405, both of which are typical Japanese HLA-DR alleles, suggesting that those T-cell epitopes might be important for the onset of house-dust mite allergy in the Japanese population. We prepared 15 analog peptides of the HLA-DRB1* 1502-restricted 15-mer peptide. Of those 15 residues, five (F35, L37, A39, F41, and E42) were critical for the epitope activity, and three residues (F35, A39, and E42) seemed to be included in anchor motifs for HLA-DRB1* 1502. The epitope peptide was also recognized by HLA-DRB1* 1502-positive healthy donors; however, only allergic T cells showed Th2 functions. Antigen-presenting cells of nonallergic donors were able to activate allergic T cells to express Th2 function. This seemed to suggest that antigen recognition of T cells, as well as additional unknown factors which promote Th2, rather than Th1, responses, might be important for the onset of house-dust mite allergy.     

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.     

Association between mite allergen (Der p 1, Der f 1, Blo t 5) levels and microscopic identification of mites or skin prick test results in asthmatic subjects

BACKGROUND: Mite allergens have been involved in airway sensitization and allergic diseases. Immunoassays for the identification and quantifiction of house dust mite (HDM) allergens are useful to improve the knowledge of regional mite fauna and the remediation of mite allergens in allergic diseases. The present study analyzed the association between levels of HDM allergen and results of mite identification or skin prick test (SPT) in two different areas of Bahia, Brazil. METHODS: Forty-two asthmatic subjects from a rural area (group I; n = 21) and a slum (group II; n = 21) were evaluated through SPT with HDM allergens and had dust samples collected at their homes for mite identification and allergen measurements. RESULTS: Positive SPT to Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis allergens were observed in 42.9, 38.0 and 42.9% subjects from group I and in 47.6, 19.0 and 33.3% subjects from group II, respectively. D. pteronyssinus and B. tropicalis were identified in approximately 76 and 50% of samples from both groups, respectively. D. farinae was identified in 38.0 and 9.5% of samples from groups I and II, respectively (p < 0.005). Der p 1, Der f 1 and Blo t 5 detection were associated with mite identification (p < 0.05). Association between HDM allergen levels over 2 microg/g of dust and positive SPT occurred only with D. pteronyssinus (p < 0.0001). CONCLUSIONS: D. pteronyssinus was the most prevalent mite species in this study followed by B. tropicalis and D. farinae. Immunoassays done to measure mite allergens were associated with mite-species identification. We conclude that these three mite species must be included on panels for the diagnosis of allergic airway diseases in subjects living in such regions.     

Epitope-specific T-cell responses and allergic phenotypes: implications for T-cell peptide therapy

In the 1990s, elucidation of the primary amino acid sequence of several major allergens using molecular cloning techniques opened the door to T-cell epitope mapping studies. Such analyses underscored the complexity of the allergen-specific T-cell repertoire and the challenges to using allergen-derived peptides to identify epitope-specific differences associated with allergic and nonallergic responses. This review highlights important factors that may influence the nature of epitope-specific T-cell responses observed in vitro. These include the properties of the allergen, genetics of the host and selection of patients with defined allergic phenotypes based on serum antibody profiles and skin test reactivity. By taking these factors into account, T-cell epitope-specific differences associated with distinct allergic phenotypes can be identified. Observations at the T-cell epitope level undermine the Th1/Th2 paradigm as a model for the development of allergic versus nonallergic responses. Instead, they support the mounting data that point to a network of interactions between T helper cells and regulatory T cells, which controls the allergic response. The ability of peptides that localize to polypeptide chain 2 of the major cat allergen, Fel d 1, to preferentially induce interleukin-10 and interferon-γ is discussed. Mechanisms whereby specific allergen-derived peptides may modify the T-cell repertoire and influence the immune outcome are also outlined. Further investigation of allergen-derived T-cell epitopes is warranted in order to optimize the design of peptide vaccines for the treatment of allergic disease.     

Inhibition Experiments with Solid Phase Mite or Epithelia in House Dust Hypersensitivity

The present study was undertaken to verify that mites are not the only allergens in house dust extracts and that other allergens such as cat epithelia can also be responsible for house dust hypersensitivities detected both by house dust skin tests and house dust RAST studies. In order to determine whether mite or epithelia fixed on a solid phase could remove not only the IgE antibodies reactive with the homologous allergens, but also the IgE antibodies reactive with house dust allergens, the authors have absorbed 10 sera of house dust allergic patients with solid phase mite or epithelia. The absorption procedure removed a large part of the IgE antibodies reactive with specific immunosorbent (Dermatophagoides pteronyssinus or cat epithelia) and in the same way the IgE antibodies reactive with house dust immunosorbent. The percentage of RAST inhibition varied from 65% to 92% for Dermatophagoides pteronyssinus and from 65% to 94% for house dust in patients allergic to house dust and mite; the percentage of RAST inhibition varied from 67% to 92% for cat epithelia and from 73% to 90% for house dust in patients allergic to house dust and cat epithelia. This is in accordance with the hypothesis that house dust is not an allergen per se, but rather a complex mosaic of several allergens including mite, animal epithelia, etc.     

HLA class II restriction specificity of Dermatophagoides spp. reactive T lymphocyte clones that support IgE synthesis

The results of recent experiments investigating the restriction specificity of cross-reactive, or Dermatophagoides farinae-specific, T cell clones isolated from an atopic individual with perennial rhinitis are reviewed. The restriction specificity was examined using serological inhibition, allogeneic presenting cells and murine fibroblasts expressing HLA-D region products. Although serological inhibition studies suggested that DR class II proteins were the major restriction elements used, the patterns of recognition observed with the allogeneic cell panel were complex, generally failing to correlate with the serologically defined MHC class II specificities. Analysis of the restriction patterns indicated that the majority of the T cell clones were restricted by DR beta III gene products and this was confirmed using murine fibroblasts expressing DRw52. DR beta I gene products functioned as restriction elements in the recognition of house dust mite allergen by the other clones. In an in-vitro model of allergen-dependent IgE synthesis, both DR beta I and DR beta III class II restricted T cells could be shown to provide functional help for IgE synthesized by autologous B cell-enriched populations.     

House dust mites in the West Indies

The purpose of this study was to determine the role of the house dust mite. Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), in allergic diseases on the island of St. Lucia. Dust samples were taken from living quarters of patients and hospital volunteers. The level of Df antigen in these living quarters was measured by RAST inhibition. All samples showed Df antigen within a large range of values. Prick tests were done to house dust mite and molds on 91 patients with suspected allergic disease with 63 (69%) reacting to at least one antigen. Of these, 56 (88.9%) reacted to one or both mites. House dust mite is a major allergen in St. Lucia.     

Population analysis of cellular responses to synthetic peptides of Der p II, a major allerg molecule of Dermatophagoides pteronyssinus, in allergic and nonallergic subjects

Okano M, Nagano T, Kino K, Yasueda H, Baba Y, Saito C, Masuda Y, Ohta N. Population analysis of cellular responses to synthetic peptides of Der p II, a major allergen molecule of Dermatophagoides pteronyssinus , in allergic and nonallergic subjects.
Responses of peripheral blood mononuclear cells to synthetic oligopeptides of Der p II, one of the major allergen molecules of Dermatophagoides pteronyssinus , were compared between allergic and nonallergic subjects. Healthy subjects showed positive responses to crude extracts of D. pteronyssinus , but only allergic subjects showed elevated cellular responses to Der p II. We synthesized three oligopeptides of Der p II in which motifs of a possible T-cell epitopc were included. Of 14 subjects with positive response to Der p II, three responded to all three peptides, while five did not respond to any peptide tested. In 11 allergic patients who showed positive response to Der p II, responsiveness to the peptide K33-T47 was significantly higher than that to other peptides ( P <0.05). All the responding patients were also positive for scratch test to Der p II, suggesting that those epitopcs induced IgE-promoting helper T-cell response in allergic persons. On the other hand, the in vitro cellular responses were not necessarily correlated to IgE production against Der p II in healthy subjects.     

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.     

Sensitization to house dust mites in Reykjavik, Iceland, in the absence of domestic exposure to mites

BACKGROUND: House dust mites are common sources of indoor allergens. In Reykjavik, Iceland, 9% of the young adult population had serum-specific IgE to Dermatophagoides pteronyssinus. Sensitization to mites is usually assumed to be due to exposure to house dust mites in the indoor environment. This investigation was carried out to measure the concentrations of house dust mite allergens and to investigate which species of mites were present in beds in Iceland. METHODS: A total of 197 randomly selected adults were visited at home using the European Community Respiratory Health Survey (ECRHS) II Indoor protocol. Dust samples were collected from mattresses for measurement of house dust mite allergen concentrations and to estimate the number and type of house dust mites. Additional samples from mattresses and floors were collected from the homes of 10 patients with positive skin prick tests (SPT) to D. pteronyssinus. House dust mite allergen concentrations were measured using ELISA and examination of mite species was carried out using microscopy. Climatic parameters were assessed using psychrometer readings in the bedrooms and outdoors. RESULTS: We found two single mite specimens, both D. pteronyssinus, in two dust samples. Mite allergen analyses indicated that two other dust samples had Der f 1 results close to the cut-off of 0.1 microg/g of dust. No samples were positive for Der p 1. In an additional collection of dust from the homes of 10 SPT-positive patients no Dermatophagoides spp. were found. CONCLUSIONS: Reykjavik citizens are exposed to extremely low amounts of house dust mite allergens in their homes. Possible alternative sources for sensitization are discussed, such as bird nests, exposure from travelling abroad, or other mites or invertebrates that cross-react with house dust mite allergens. Our findings suggest that exposures other than to house dust mites indoors are possible sources of mite allergen exposure.     

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.     

Cloned human T lymphocytes reactive with Dermatophagoides farinae (house dust mite): a comparison of T- and B-cell antigen recognition.

In this report, T-cell and B-cell recognition of the house dust mite Dermatophagoides farinae (D. far.) is compared. Nitrocellulose immunoblots of polyacrylamide gel electrophoresis (SDS-PAGE)-fractionated D. far. were added to proliferation assays to map the antigen specificity of cloned human helper T cells and a long-term line induced with D. far. T-cell recognition was of a polypeptide of molecular weight 9000-13,000, that migrates with the serologically defined allergen Der fII (12,500 MW). Since the cloned T cells, unlike the polyclonal response, failed to respond to Dermatophagoides pteronyssinus (D. pter.), this suggests that they recognize a species-specific epitope. In contrast, analysis of the B-cell response using Western blotting demonstrated that, in addition to Der fII, antibodies reactive with the major allergens Der fI (26,000 MW) and Der fIII (29,000 MW) were present in the serum. Similar specificities were seen in the antibody response to D. pter., and while it has been reported that the B-cell response to D. far. and D. pter. are predominantly cross-reactive, our observations suggest that species-specific CD4-positive T cells are present in the overall cellular response to D. far.     

Discrepancies between in vitro and in vivo tests for house dust mite allergy: is domestic exposure a better predictor than sensitization?

We subjected seven asthmatic children to two bronchial allergen challenges, first with an extract from the house dust mite Dermatophagoides pteronyssinus (Derp) and then Dermatophagoides farinae (Der f), or vice versa. All children had elevated specific serum IgE to both species as well as reactions by crossed radioimmuno/electrophoresis (CRIE) to both group I and II allergens from both species. Immunoabsorption and subsequent analysis by CRIE showed a considerable concentration of serum IgE with specificity for epitopes common to the two species of house dust mite. Home dust sampling established that all children were exposed to Der f and only two to Der p. On bronchial provocation tests, all responded to Der f with an immediate reaction and five with a late reaction, only three of seven showed an immediate response to Der p, with four of the seven showing a late reaction. Our data could indicate that the local allergic immune reaction in the respiratory tract is sustained by ongoing exposure, and may thus have a different species specificity than the response reflected in the serum. In conclusion, our data indicates a lack of association between in vitro and in vivo tests for house dust mite allergy, which supports the continuing need for monitoring current clinical sensitization by allergen provocation tests and by measuring domestic exposure to the corresponding allergen. Extended studies are needed to support our findings.     

Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes

The contribution of the HLA-DRB1, -B3, and -B5 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatible and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Oligonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2DW21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR beta chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of allergic inflammation in the lungs of sensitized sheep after local challenge with house dust mite

Background Previous sheep models of asthma are based on sheep sensitized to nematode (Ascaris) allergens and these have been used to evaluate the physiological and pharmacological effects of potential anti‐asthma agents. The immunological mechanisms associated with the allergic response in sheep lungs has not been examined in detail. Objective To develop an experimental sheep model of allergic lung inflammation based on a relevant major human allergen, house dust mite, and to define the immunological features of the allergic response in this model. Methods Sheep immunized subcutaneously with solubilized house dust mite extract were given a single bronchial challenge with house dust mite. Bronchoalveolar lavage (BAL) and peripheral blood leucocytes were collected before and after challenge for flow cytometry, and tissue samples were taken post‐mortem (48 h post‐challenge) for histology and immunohistochemical analyses. Results Immunizations with 50 μg house dust mite induced an allergen‐specific IgE response in 50 to 60% of sheep (allergic sheep), with higher antigen doses increasing specific IgG₁ but not IgE. Lung challenge of allergic sheep with house dust mite led to the initial recruitment of neutrophils (at 6 h post‐challenge) followed by eosinophils and activated lymphocytes into the lung tissue and BAL, similar to the late‐phase allergic response seen in human asthma. Eosinophil recruitment peaked at 48 h post‐challenge, representing 10 to 33% of BAL leucocytes in allergen‐challenged allergic sheep compared to 0 to 3% in allergen‐challenged control (naïve) sheep. Lymphocytes recovered from the lung after allergen challenge were enriched for CD4⁺ T cells and were more activated than lymphocytes in blood. There was significant down‐regulation of CD62L (L‐selectin) and CD49d (VLA‐4) expression after allergen challenge on BAL eosinophils and lymphocytes compared to blood. In addition, VCAM‐1 (ligand for VLA‐4) was up‐regulated on blood vessels of allergen‐challenged lungs. Eosinophils, CD4⁺ T cells and CD45R⁺ B cells were the most prominent leucocytes found in lung tissue 48 h after allergen challenge. Conclusion This study demonstrates, for the first time, the ability of house dust mite to induce allergic responses in sheep lungs. This novel sheep model of allergic lung inflammation using relevant human allergens, exhibits similarities to human asthmatic disease and will be a useful tool for studies of the immunological and physiological mechanisms of allergic asthma.     

Allergenicity and physicochemical characterization of house dust mite derived amylase.

The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus. In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I. Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000. The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGXRSVITXLME. It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic. The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds.