IgE responses to Dermatophagoides pteronyssinus native major allergens Der p 1 and Der p 2 during long-term specific immunotherapy

We investigated by ELISA the IgE response to whole extract of the house-dust mite Dermatophagoides pteronyssinus (Dp) and to the native major allergens, Der p 1 and Der p 2, in sera from 18 adult patients (group A) with Dp-allergic asthma before ( t ₀) and 1, 2, 3, and 4 ( t ₁– t ₄) years after subcutaneous specific immunotherapy (SIT). A qualitative reduction ( P =0.05) of the IgE responses to Dp and Der p 2 was observed from t ₁ to t ₄, but a highly statistical significant decrease appeared at t ₃, ( P < 0.01). With regard to Der p 1 IgE values, the immunotherapy induced a significant decrease ( P < 0.01) at t ₃, but not before. In group A, the IgE responses to Der p 1 and Der p 2 were not correlated at t ₀ ( r _s=0.31; P = 0.2l) but were correlated at t₃ ( r _s= 0.78; P=0.001). We also examined sera from 14 adult patients (group B, same SIT schedule as group A) who were without respiratory symptoms at the end of the third year (t₃) of Dp SIT. At this time ( t ₃), there were no significant differences in Der p 1 and Der p 2 IgE levels between group A and group B.     

Cytokine responses to Der p 1 and Der p 7: house dust mite allergens with different IgE-binding activities

BACKGROUND: There is very limited information comparing T-cell responses to different house dust mite (HDM) allergens even though T cells are essential in the initiation and regulation of immunoglobulin (Ig) E synthesis and eosinophilia. OBJECTIVE: To compare the level of T-cell proliferation and cytokine production to the group 1 and group 7 HDM allergens which are known to have different IgE-binding capabilities. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMC) from HDM-allergic and HDM-nonallergic donors were stimulated with the group 1 and group 7 allergens of Dermatophagoides pteronyssinus and the level of proliferation as well as IL-5 and IFNgamma production were measured. RESULTS: The proliferative and IL-5 production to the group 1 and group 7 allergens were equivalent despite the group 7 allergen's lower frequency of IgE-binding. However more IFNgamma was produced to Der p 7 than to Der p 1, particularly for the nonallergic donors. As expected IL-5 production was much higher for PBMC from the allergic donors than for cells from nonallergics; however, there was no difference in the level of T-cell proliferation between the donor groups. CONCLUSION: The relative importance of the individual HDM allergens is normally determined by measuring the frequency of IgE-binding to the allergen in sera from an allergic population. The equivalent increased IL-5 response of PBMC from allergic people to the group 1 and group 7 allergens despite the different IgE-inducing activity indicates that these allergens may be equally capable of contributing to an asthmatic response by inducing eosinophilia.     

Inhibition of mucosal and systemic T(h)2-type immune responses by intranasal peptides containing a dominant T cell epitope of the allergen Der p 1

Although the intranasal administration of peptides containing T cell epitopes has been shown to be a potent method of inhibiting responses to the allergen Der p 1, the experiments to date have concentrated on their ability to regulate immune responses to the injection of antigen in a T(h)1-type adjuvant. Their ability to regulate responses to a T(h)2-type immunization and to sensitization via the respiratory mucosa has not been examined. Here it is shown that peptide used in doses required to block delayed-type hypersensitivity can also readily inhibit IgE responses to Der p 1 injected in alum. To examine responses induced in the respiratory mucosa, mice pretreated with intranasal peptide were sensitized with an intranasal dose of Der p 1 in conjunction with a mutated enterotoxin adjuvant. Intranasal peptide even in very high doses did not reduce IgE titers, but the ability of cells from the draining lymph nodes to release IL-4 and IL-13 but not IL-2, IL-5, IL-10 or IFN-gamma was reduced. These are the first reports on the effect of intranasal peptides containing T cell epitopes on IgE in T(h)2 immunization and on responses to respiratory immunization. Thus the effect of the peptide-induced mucosal tolerance differs depending on the type of immunization used for sensitization, but the potential to inhibit T(h)2 responses and responses to respiratory sensitization as well as T(h)1 responses has been demonstrated.     

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris

BACKGROUND: The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. OBJECTIVE: The aim of this study was to establish an efficient system for the production of recombinant Der p 1. METHODS: The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. RESULTS: ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. CONCLUSION: This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.     

Diagnostic significance of in vitro T-cell proliferative responses to house-dust mite Der p 1 in children with dust-mite allerev

The study aimed to investigate the possible diagnostic significance of T-cell proliferative responses to purified Der p 1 antigen in allergic children with and without allergic sensitization to house-dust-mite (HDM) allergens. T-cell reactivity to purified Der p 1antigen was analyzed in 24 children with allergic sensitization to HDM demonstrated by RAST and/or positive skin prick tests. Twelve healthy young adults and 11 children with allergic sensitizations other than HDM served as controls. Of 25 HDM-allergic patients, 16 displayed strong T-cell reactivity to Der p 1 (stimulation indices >3): nine patients showed no T-cell proliferation despite the presence of specific IgE, and five showed no responses despite positive skin prick test. In two patients, a weak T-cell response to Der p 1 could be demonstrated in the absence of specific IgE and negative skin test result. The two affected subjects did not show evidence of mite allergy. No T-cell responses were observed in adult controls (stimulation indices<3). We conclude that the assessment of T-cell reactivity to Der p 1 is of little value for the diagnosis of HDM allergy. The importance of T-cell proliferative responses for the study of the pathogenesis of HDM allergy remains unchallenged.     

Genetic variation of Der p 2 allergens: effects on T cell responses and immunoglobulin E binding

BACKGROUND: Der p 2 is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variations on T cell and antibody responses has not been compared. OBJECTIVES: To compare IgE antibody binding and T cell proliferation and cytokine release induced by variants of Der p 2. METHODS: Peripheral blood mononuclear cells (PBMC) from 19 allergic and 15 non-allergic people were stimulated with recombinant variants of Der p 2. IL-5, IL-10, IL-13 and IFN-gamma were measured by a time resolved fluorescence (TRF) assay. Serum IgE antibody was measured using a solid-phase TRF assay. RESULTS: Overall the most prevalent variant of Der p 2 (Der p 2. 0101) was the highest or approximately equal highest inducer of T cell proliferation and IL-5, IL-10, IL-13 and IFN-gamma release. The most divergent variant 0104 induced the next highest responses. The variants 0107 and 0108 showed interesting changes especially when the allergic status was considered. Responses to 0107 showed poor Th1/Th2 polarization and, except for IL-10 release, cytokine responses to 0108 were low for non-allergic subjects. The variant 0101 showed similar monoclonal antibody binding but moderately less IgE binding than the other variants. CONCLUSIONS: The most prevalent variant, Der p 2. 0101, was the most active for T cell stimulation and although its IgE binding was slightly less than other variants that was highly correlated. The variant Der p 2. 0104 which contains the known common polymorphic changes had a response which was similar to Der p 2. 0101 and thus these two variants were the most stimulatory representations of Der p 2. The T cell responses to the less common variants 0107 and 0108 however, showed consistent differences demonstrating that changes in the sequence could change the cytokine response.     

The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level

BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN-gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development.     

Use of a chimeric ELISA to investigate immunoglobulin E antibody responses to Der p 1 and Der p 2 in mite-allergic patients with asthma, wheezing and/or rhinitis

BACKGROUND: Sensitization to indoor allergens, particularly to dust mites, is a strong risk factor for asthma in children and adults. Assessment of sensitization is carried out using in vivo and in vitro tests to detect specific IgE antibodies. OBJECTIVE: To investigate IgE antibody responses to mites in patients with asthma, wheezing and/or rhinitis, using chimeric ELISA to measure specific IgE antibodies to mite allergens Der p 1 and Der p 2. METHODS: Specific IgE antibodies to Der p 1 and Der p 2 were quantified by chimeric ELISA, and compared with IgE to Dermatophagoides pteronyssinus (Dpt) measured using the CAP system (Pharmacia). A panel of sera from 212 patients with asthma, wheezing and/or rhinitis and 11 controls was analysed. RESULTS: There was a significant correlation between IgE to Dpt measured by CAP and IgE to Der p 1 (r = 0.81, P < 0.001), Der p 2 (r = 0.79, P < 0.001) and combined Der p 1 and Der p 2 (r = 0.86, P < 0.001). Seventy per cent of all patients had IgE to Dpt, and of those, 76.5% had IgE to Der p 1, 79.2% had IgE to Der p 2 and 83.1% had IgE to Der p 1 and Der p 2 combined. Considering the cut-off level of 2 IU/mL of IgE to either Der p 1 or Der p 2, the predictive value for a positive IgE to Dpt by CAP was greater than 95%. CONCLUSIONS: The chimeric ELISA allowed accurate quantification of IgE antibodies to Dpt allergens Der p 1 and Der p 2, and it could be useful for studying immune responses to mites in patients with asthma and/or rhinitis.     

Stimulatory and inhibitory epitopes in the T cell responses of mice to Der p 1

BACKGROUND: The responses of mice to the mite allergen Der p 1 have been used to study the mechanisms of allergic sensitization and the development of new types of immunotherapy. Many of the studies require a knowledge of the T cell epitopes, and because Der p 1 is polymorphic, the effect of natural amino acid substitution in the allergen. The intranasal administration of peptides containing T cell epitopes can induce a mucosal tolerance but it is not known if the major activity is limited to stimulatory peptides and if, as found for autoimmunity, some epitopes are not inhibitory. OBJECTIVE: To determine and compare the sequences of Der p 1 which contain stimulatory epitopes for the high responding H-2(b) and H-2(q) mice and the sequences which induce tolerance by intranasal administration of peptides. METHODS: T cell responses of mice immunized with Der p 1 were measured by in vitro T cell stimulation assays so an extensive study of epitope recognition and intranasal tolerance could be made. Synthetic peptides were used to examine the stimulatory and inhibitory ability of all Der p 1 sequences and to map the major H-2(b) epitope in detail. This included the effect of the common polymorphic amino acid 124 substitution found within this epitope. RESULTS: Three and two regions, respectively, were found to contain stimulatory T cell epitopes for H-2(b) and H-2(q) mice. The peptides in these regions were also the most active at inducing intranasal tolerance for the responding haplotype. The correspondence between inhibitory and stimulatory peptides was maintained for the fine mapping of the major H-2(b) epitope. This was found about a core region of 118-126 which was overlapping but separate to a consensus sequence for the binding of endogeneous peptides. Peptides with alanine at the naturally polymorphic residue 124 stimulated and inhibited responses to Der p 1 more effectively, while peptides with the valine 124 variant were immunogenic but poorly cross-reactive. CONCLUSIONS: The intranasal administration of peptides representing each of five epitopes recognized by two strains of mice were able to induce mucosal tolerance and the major tolerizing activity was limited to these epitopes. The position of the core major epitope for C57 mice, which differs from a previously predicted epitope, and its specificity for the natural alanine 124 variant is described.     

The proteolytic activity of the major dust mite allergen Der p 1 enhances the IgE antibody response to a bystander antigen

BACKGROUND: We have recently demonstrated that immunization of mice with proteolytically active Der p 1, the major dust mite allergen, results in a significant enhancement in total and Der p 1-specific IgE synthesis compared to mice immunized with Der p 1 that has been irreversibly blocked with the cysteine protease inhibitors E-64 and iodoacetamide. Thus, the demonstration that the proteolytic activity of Der p 1 enhances total IgE production, apart from increasing Der p 1-specific IgE, suggests that this allergen may have an IgE-specific adjuvant effect. OBJECTIVE: To determine if the proteolytic activity of Der p 1 has an IgE-specific adjuvant effect. METHODS: We have examined this concept in experiments whereby ovalbumin, used as a bystander antigen, was injected alone or coinjected with either proteolytically active or inactive Der p 1 into groups of mice and IgE and IgG antibody responses were measured. RESULTS: Here we demonstrate for the first time that the proteolytic activity of Der p 1, when given at 10-fold higher concentration, enhances the IgE antibody response to ovalbumin. CONCLUSIONS: These findings show that the proteolytic activity of Der p 1 leads to the augmentation of IgE antibody responses to itself and to other allergens present in the microenvironment.     

Human T cells that have been conditioned by the proteolytic activity of the major dust mite allergen Der p 1 trigger enhanced immunoglobulin E synthesis by B cells

BACKGROUND: We have previously demonstrated that the proteolytic activity of Der p 1 selectively cleaves human CD25, the 55 kDa alpha subunit of the IL-2 receptor. As a result of cleavage of surface CD25, peripheral blood T cells produce less IFN-gamma and more IL-4, thereby leading to progressive polarization of the T cells towards a Th2 cytokine profile. Therefore, these observations underline the potential role of the proteolytic activity of Der p 1 in creating a microenvironment conducive for IgE synthesis. OBJECTIVE: To study the effect of T cells that have been conditioned by the proteolytic activity of Der p 1 on IgE synthesis by B cells. METHODS: We have examined this concept in experiments whereby T cells that have been exposed to either proteolytically active or inactive Der p 1 were cocultured with autologous B cells and IgE antibody synthesis was monitored. RESULTS: Here we demonstrate for the first time that coculturing T cells that have been in contact with proteolytically active Der p 1 with autologous B cells leads to augmentation of IgE antibody responses. CONCLUSIONS: The proteolytic activity of Der p 1 conditions human T cells, which then become empowered to trigger enhanced IgE synthesis by B cells.     

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.     

T cell cytokine responses to outer membrane proteins of Haemophilus influenzae and the house dust mite allergens Der p 1 in allergic and non-allergic subjects

BACKGROUND: Haemophilus influenzae are ubiquitous colonizers of the nasopharynx, Little is known about the T cell cytokine responses to the antigens of these bacteria and whether or not the responses may interact with responses to aeroallergen. OBJECTIVE: To measure the T cell cytokine responses to conserved outer membrane protein antigens of Haemophilus influenzae and to house dust mite allergen of subjects allergic to the house dust mite and of subjects without allergic sensitization. METHODS: T cell responses were measured by in vitro proliferation and cytokine release from peripheral blood monocytes (PBMC). The allergen used was Der p 1 and outer membrane proteins were recombinant polypeptides representing the OMP6 and D15 antigens. RESULTS: The PBMC of most subjects had proliferative responses to OMP6 and D15, which were highly correlated. The pattern of cytokine release was Th1 biased with high levels of IFN-gamma and usually little IL-5 or IL-13 although PBMC from a few subjects did release IL-5 independent of allergic status. IL-10 release was readily detected. There was no difference in the anti-OMP cytokine response of PBMC from subjects without any known allergy and the responses of PBMC from subjects who were highly allergic to house dust mite. The responses to the Der p 1 allergen showed the expected Th2 cytokine release. CONCLUSION: The outer membrane protein antigens of the ubiquitous colonizing bacteria Haemophilus influenzae induce Th1 cytokine responses which are similar for PBMC from non-allergic individuals and subjects with a high degree of allergy to the perennial house dust mite allergen and strong Th2 responses to Der p 1.     

Indoor determinants of Der p 1 and Der f 1 concentrations in house dust are different

BACKGROUND: Exposure to mite allergens is a major risk factor for sensitization and the development of asthma. Der p 1 and Der f 1 content in homes and probably the proportion of both antigens is highly variable even in the same geographical area. OBJECTIVE: We investigated specific indoor determinants of Der p 1 and Der f 1 concentrations in house dust of two German cities, Erfurt and Hamburg (n = 405 homes). METHODS: Mite allergen levels were determined using monoclonal antibodies against Der p 1 and Der f 1 by the ELISA method. Indoor relative humidity and temperature were monitored continuously in the homes over 1 week. The characteristics of homes and occupants were assessed by questionnaire to obtain information on factors which may have an impact on the mite antigen concentration in house dust. These determinants were studied by multivariate regression analysis. RESULTS: The correlation between concentrations of Der p 1 and Der f 1 inside the homes was weak (r = 0.29-0.35), indicating that different determinants are relevant. Concentrations of the allergens were significantly higher on lower floors (ratios 2-8 times, Der p 1, Der f 1), on old mattresses (ratios 3-13 times, Der p 1, Der f 1), in post-war buildings (ratio 6 times, Der p 1), for non-central heating (ratio 2 times, Der p 1), for old carpets (ratio 3 times, Der p 1) and for the presence of a dog in the house (ratio 3 times, Der f 1). Furthermore, mite concentration increases with raising relative humidity (ratio 1.03 per 1%, Der p 1) and with decreasing temperature (ratio 0.86 per 1 degrees C, Der p 1) indoors. CONCLUSION: Both Der p 1 and Der f 1 concentrations should be measured in house dust, since they are only weakly correlated and have different determinants.     

House dust mite allergen (Der p 1) accumulation on new synthetic and feather pillows

BACKGROUND: We have previously demonstrated that synthetic pillows contain significantly more Der p 1 than feather pillows. The aim of this study was to compare the accumulation of Der p 1 allergen on new synthetic and new feather pillows. METHODS: Der p 1 was measured in dust samples from pairs of synthetic and feather pillows placed together on 12 beds over a 12-month period. RESULTS: After 12 months synthetic pillows contained higher concentrations of Der p 1 (19.28 microg/g; 95% confidence interval: 9.76-38.07) than feather pillows (6.45 microg/g; 2.96-14.05). There was a significant correlation between Der p 1 concentrations of pillows at 12 months and Der p 1 concentrations of the mattresses at the beginning of the study (r = 0.72; P = 0.008 for both types of pillows). CONCLUSIONS: Synthetic pillows accumulate Der p 1 more rapidly than feather pillows and the accumulation rate of Der p 1 on pillows is governed by the Der p 1 concentration in the immediate environment they are placed in.     

Long-range destruction of Der p 1 using experimental and commercially available ionizers

BACKGROUND: To reduce the risk of sensitization and the elicitation of allergy symptoms, it is important to reduce the level of allergens in the home. It has previously been demonstrated that corona discharge, the process by which ionizers produce ions, can destroy the major house dust mite allergen Der p 1. OBJECTIVE: In this paper the denaturing efficacy of an experimental ionizer and two commercially available products are evaluated. METHODS: The first test was conducted in an electrically grounded chamber with samples of Der p 1 placed in various positions for 1, 2 and 3 weeks. The second test was conducted in situ in an unoccupied, furnished office room for 1 week. Der p 1 concentration was quantified by two-site monoclonal antibody enzyme-linked immunosorbent assay (ELISA). RESULTS: All ionizers in both tests caused significant reductions in allergen concentration (P < 0.05), reaching a maximum of 92% with the experimental ionizer in the chamber after 3 weeks. The percentage reductions observed in situ with the experimental and the larger commercial ionizer were similar, reaching a maximum of 32% at a distance of 4 m away from the experimental ionizer after 1 week of exposure. CONCLUSION: With a revised protocol for use, air ionizers may offer a simple, efficient and inexpensive way to reduce allergen levels in the domestic environment.     

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.