Absence of continuous epitopes in the house dust mite major allergens Der p I from Dermatophagoides pteronyssinus and Der f I from Dermatophagoides farinae

Background The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes.
Objective The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic detertninants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences.
Methods In order to identity any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs.
Results None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs.
Conclusion The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.     

The late asthmatic response is associated with baseline allergen-specific proliferative responsiveness of peripheral T lymphocytes in vitro and serum interleukin-5

BACKGROUND: Increasing insights into the mechanism underlying the allergen-induced late asthmatic response (LAR) have been gained with implication of activated eosinophils and CD4+ T lymphocytes. However, the patient characteristics that indicate the individual capacity to develop a LAR are not well-defined. METHODS: In 22 subjects with mild to moderate house dust mite-allergic asthma, we investigated the relationship between the LAR and two other models of late-phase allergic inflammation, i.e. the allergen-specific proliferative response of peripheral blood T lymphocytes in vitro and the late cutaneous response. Non-specific bronchial responsiveness (PC20histamine), lung function (FEV1), peripheral blood eosinophil count, early phase allergic skin sensitivity, and levels of total and specific immunoglobulin E (IgE) were determined prior to bronchial allergen challenge. Serum levels of interleukin-5 (IL-5) were measured before and at several time points after allergen inhalation. RESULTS: A significant correlation was found between the magnitude of the LAR and the allergen-specific proliferative response of peripheral T lymphocytes (r = 0.44, P = 0.04) but not the late cutaneous response. Stepwise-multiple linear regression of the magnitude of the LAR on the parameters analysed at baseline, resulted in a model combining PC20 histamine, early phase allergic skin sensitivity, and the allergen-specific proliferative response of peripheral T lymphocytes (R2 = 0.84, P<0.001). No contribution of the late cutaneous response to the prediction of the LAR was found. Serum levels of IL-5 increased significantly at 6 h (P = 0.01) and 24 h (P = 0.003) after bronchial allergen challenge and correlated with the allergen-specific proliferative response of peripheral T lymphocytes in vitro (rho = 0.48, P = 0.02). CONCLUSIONS: The findings in this study point to a role of TH2-lymphocyte responses in the development of the allergen-induced LAR. In allergic asthmatic patients, allergen-specific responsiveness of peripheral T-lymphocytes in vitro may serve as a model to determine the individual capacity to develop a LAR after allergen inhalation.     

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.     

Frequency and intensity of late asthmatic reactions after bronchial allergen challenge in asthma and rhinitis

The occurrence of late asthmatic reactions after bronchial allergen challenge was studied in 50 house dust mite allergic patients subdivided in three groups: one group had asthma without nasal symptoms, another group had rhinitis without pulmonary symptoms and a third group had a combination of both asthma and rhinitis. Late asthmatic reactions were present in 80% of asthmatic patients and in 18.7% of rhinitis patients. The degree of non-specific bronchial reactivity to histamine (provocative dose 15 or PD15 histamine) and the degree of immediate reactivity to allergen (PD15 house dust mite) did not differ significantly between patients with and without late asthmatic reactions. These findings suggest that an important difference between asthma and rhinitis is the lack of late asthmatic reactions in rhinitis patients, whereas the degree of immediate bronchial reactivity to the allergen is similar in asthma and rhinitis.     

Localization of a major allergen, Der p 2, in the gut and faecal pellets of Dermatophagoides pteronyssinus

BACKGROUND: The house dust mite Dermatophagoides ptronyssinus is one of the most significant indoor sensitizing agents of allergy. Allergen localization may indicate the importance of secreted materials, faeces, and nonexcreted mite body components as allergen sources. OBJECTIVE: This study attempted to localize the sites and concentrations of Der p 2 in the cryostat sections of D. pteronyssinus using antirecombinant Der p 2 monoclonal antibody. METHODS: Male and female mites and mite faeces collected separately from both sexes were used. Live mites were embedded and serial cryostat sections for light microscopy were performed. Anti-recombinant Der p 2 monoclonal antibody previously produced by the authors was used. For immunoprobing, mite cryostat sections were incubated in the following antibody-containing solutions: monoclonal antibody against Der p 2 was initially applied to the sections and fluorescent isothiocyanate conjugated antimouse immunoglobulin G was reacted as the secondary antibody. The faecal pellets were treated the same as described above. RESULTS: Immunofluorescent probing of cryostat sections with the monoclonal antibody showed labelling of the gut lining, gut contents and defecated faecal pellets. No other internal organs were identified as positively labelled. CONCLUSION: This study suggested that a major allergen, Der p 2, found in the house dust mite D. pteronyssinus is derived from the digestive tract and concentrated in the faeces.     

Mite (Der p 1, Der f 1) and cat (Fel d 1) allergens in the homes of babies with a family history of allergy

Carpet and floor dust samples were collected in four different seasons, from 39 Swedish homes of babies with a family history of allergy. House-dust mite ( Der p 1, Der f 1 ) and cat ( Fel d 1 ) allergen contents were determined by mab ELISA, and the levels were related to various environmental factors. Both mite and cat antigens were detected in 94% of the samples and in all homes, but the levels were low ( Der p 1 , range 15 ng–1944 ng/g fine dust; Der f 1 , range 14 ng–264 ng/g of fine dust; Fel d 1 , range 16 ng–3120 ng/g fine dust). Mite-allergen levels were significantly higher ( P <0.001) in floor dust than in carpets, and D. pteronyssinus predominated. In contrast, the levels of cat antigen were significantly ( P <0.05) higher in carpets than in floor dust. There was no clear relation between mite-allergen levels and type of house, except that the higher values were found in homes with dampness problems. Cat-allergen levels were higher than total mite-allergen content, and the highest levels were found in homes with a cat ( P <0.05). Rather high concentrations of cat allergen were also found in homes without a cat, which may explain why cat sensitization is so common in Sweden. As the prevalence of house-dust mite sensitivity is increasing in Swedish children, and as the individual patient threshold for eliciting symptoms varies, we suggest that sensitization may possibly occur at a lower exposure level than generally accepted as risk level for sensitization (2 μg/g dust).     

Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model

Background

Allergen‐specific immunotherapy can induce long‐term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as β‐glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment.

Aims

Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)‐driven mouse model of allergic asthma.

Materials and methods

HDM‐sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen‐induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue.

Results

ESR measurement shows suppression of early allergic response in HDM‐SCIT– and DerP1/2‐SCIT–treated mice. Both HDM‐SCIT and DerP1/2‐SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2‐SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2‐SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL‐33, CCL17 and eotaxin levels in lung tissue.

Discussion

Taken together, these data show that purified DerP1/2‐SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM‐induced activation of lung structural cells including airway epithelium.

Conclusions

We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.     

Effect of cetirizine, a new H1 antihistamine, on the early and late allergic reactions in a bronchial provocation test with allergen

Background: Cetirizine is a highly sensitive H₁ antihistamine with particular antiallergic properties, which has been shown to be effective in the treatment of allergic rhinitis, urticaria, and hay-fever-associated asthma.Methods: To assess the effect of cetirizine on the late allergic reaction to a specific bronchial provocation test (BPT) with allergen, we selected 25 patients with allergic asthma as determined by history, skin tests, and specific IgE levels. They were challenged with increasing doses of a cat or mite extract until a 20% drop in forced expiratory volume in 1 second (FEV₁) was recorded. Sixteen patients (11 men and 5 women with a mean age of 22 years; range, 18 to 48 years) exhibited a dual response (early and late allergic reactions). These 16 patients underwent a second BPT 2 weeks later, and each again showed a dual response. They were then randomized to receive either a placebo (8 patients) or cetirizine, 15 mg twice daily (8 patients) in a double-blind fashion. After 7 days of treatment, they underwent a third BPT with the same allergen dose as given in the second BPT.Results: The intensity and duration of early allergic reaction were not affected by cetirizine, whereas all parameters of late allergic reaction were statistically significantly improved in the cetirizine group when compared with those of the placebo group (maximum FEV₁ decrease, p = 0.046; FEV₁ [area above the curve], p = 0.027; maximum airway resistance increase, p = 0.021; airway resistance [area under the curve], p = 0.036).Conclusions: Cetirizine produced a significant protective effect against an allergen-induced late allergic reaction in a BPT. Cetirizine might therefore be effective in the treatment of asthma.     

Effects of house dust mite avoidance measures on Der p 1 concentrations and clinical condition of mild adult house dust mite-allergic asthmatic patients, using no inhaled steroids.

BACKGROUND: Exposure to house dust mite (HDM) allergens often results in worsening of asthma. Therefore, avoidance of exposure to HDM allergens is often proposed. Unfortunately, the most effective and feasible avoidance strategy is still not completely assessed. Consequently, we investigated the effects of a combined HDM avoidance strategy on HDM allergen concentrations and clinical condition of allergic, mild asthmatic, patients using no inhaled steroids. METHODS: Asthmatic patients, allergic to HDM, using no inhaled corticosteroids, were randomly allocated to an active (n = 76) or a placebo allergen-avoidance group (n = 81). Avoidance measures consisted of applying Acarosan(R) (placebo: water) to the living room and bedroom floors, and the use of HDM-impermeable covers for mattresses and bedding (placebo: cotton covers for mattresses only). Effects on allergen concentrations (Der p 1), FEV1, bronchial hyperresponsiveness, peak flow parameters and asthma symptom scores were studied during 20 weeks and controlled for the allergic status of the patients. RESULTS: The active covers reduced Der p 1 concentrations to 9.4% (P = 0.0001), and were always significant lower than in the placebo group (P = 0.0002). Acarosan(R) resulted in slight but significant decreases (twofold, P = 0.0001), both on living room and bedroom floors, but concentrations were never significantly lower than the placebo group. Although the combined avoidance strategy resulted in a considerable reduction in allergen load in the active group, no differences were seen between the two groups in any of the clinical parameters during the follow-up period in this group of allergic asthmatics, using no inhaled corticosteroids. Corrections for the allergic status did not alter these results. CONCLUSIONS: The combined avoidance strategy was effective in reducing HDM allergen concentration. This was especially achieved by the allergen-impermeable covers, while the effects of Acarosan(R) were only marginal. However, this allergen reduction was not reflected in a convincing improvement in clinical condition in this group of mild allergic asthmatics, using no inhaled steroids. Perhaps, a longer follow-up period would have resulted in more pronounced effects.     

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.