Dermatophagoides Pteronyssinus Extract Research Articles

The efficacy and safety of two commercial house dust mite extracts for allergic rhinitis: a head-to-head study.

Several studies have demonstrated the efficacy of house dust mite (HDM) immunotherapy in allergic rhinitis (AR). We aimed to compare the efficacy and safety of 2 commercial HDM extracts in a Chinese AR population. This was an open-label study. HDM-associated AR patients were randomized into Dermatophagoides pteronyssinus (Dp) extracts (Alutard SQ; ALK, Hørsholm, Denmark) and Dp/Dermatophagoides farinae (Df) extracts (NovoHelisen Depot [NHD]; Allergopharma, Reinbek, Germany) groups. All patients received subcutaneous injections for 1 year, and were followed every 3 months during that 1-year period. Symptom score, medication score, and adverse reactions were recorded. The primary endpoint was the total combined symptom and medication score (CSMS) during the efficacy evaluation period. Blood samples were taken for specific immunoglobulin E (IgE), IgG4, and IgE-blocking factor tests at baseline and after the 1-year treatment. A total of 230 AR patients were randomized; 29 patients dropped out. Analysis of the primary endpoint demonstrated significant reductions in CSMS of 1.8 vs 3.1 (p < 0.001) in the Alutard group and 1.8 vs 3.3 (p < 0.001) in the NHD group compared with baseline. The 2 groups presented equal effectiveness with regard to CSMS, symptom score, and medication score (p > 0.05). The treatment was well tolerated in both groups; 17 (14.8%) patients experienced systemic reactions (SRs) in the Alutard group and 13 (11.3%) in the NHD group. The rates of SRs showed no difference in the 2 groups (p > 0.05), and no anaphylaxis occurred. IgG4 and IgE-blocking factor to Dp and Df were increased significantly in both groups after the 1-year treatment. Our study confirmed the equal efficacy and safety profile of both commercial extracts in HDM-associated AR patients.

Evaluation of major mite allergens from European standardized commercial extracts for in vivo diagnosis: addressing the need for precision medicine

BackgroundSkin prick testing is the first-line interventional method to diagnose IgE mediated allergic diseases. Methodological differences in manufacturing processes and extract standardization may lead to variations in the reagent quality and potency. The current study evaluates sixteen commercially available Dermatophagoides pteronyssinus and Blomia tropicalis extracts for allergy diagnosis from different European manufacturers regarding allergen composition and content and whether these differences could influence their biological activity.MethodsMite-allergic subjects (n = 21) were skin-tested with the extracts and studied for immunoglobulin E reactivity. Nine extracts from D. pteronyssinus and seven from B. tropicalis were analysed for total protein content by Bradford and ELISA double sandwich was used to quantify specific antibodies for D. pteronyssinus and B. tropicalis major allergens from nine different manufacturers.ResultsMite extracts showed a 10–60 fold variation regarding the total protein content. The contents of the major allergens of D. pteronyssinus and B. tropicalis differed considerably (30–53 fold change) among the extracts. Blo t 5 was quantitatively present in < 50% of the of the B. tropicalis reagents and could not be clearly detected by immunoblotting in the majority of the B. tropicalis commercial extracts.ConclusionsCertain natural D. pteronyssinus and B. tropicalis extracts lack important allergens showing a considerable variability in composition and content. A closer collaboration among clinicians, allergen manufacturing companies and regulatory agencies to improve the quality and consistency of D. pteronyssinus and B. tropicalis extracts is warranted to achieve a more precise diagnosis and treatment of house dust mite allergy.

Altered Sphingolipid Metabolism Is Associated With Asthma Phenotype in House Dust Mite-Allergic Patients.

PurposeSphingolipids play an important role in cell growth, survival, inflammation and tissue remodeling. House dust mite (HDM) allergy is a major risk factor for asthma. The aim of the study was to evaluate if allergic asthma phenotype is associated with altered sphingolipid metabolism.MethodsTwenty-two HDM-allergic asthmatic patients and 11 HDM-allergic rhinitis patients were challenged intrabronchially with biologically standardized Dermatophagoides pteronyssinus extract. Whole blood and platelet-poor plasma samples were collected before, during early asthmatic response (EAR), late asthmatic response (LAR) and 24 hours after the challenge. Concentrations of sphinganine (SFA), sphinganine-1-phosphate (SFA1P), ceramide, sphingosine (SFO) and sphingosine-1-phosphate (S1P) were measured using high performance liquid chromatography.ResultsIn all house dust mite-allergic patients (HDM-APs), baseline lung function and severity of airway hyperreactivity (AHR) correlated significantly with plasma S1P and SFA1P concentrations. Exhaled nitric oxide concentration, however, correlated with SFA and ceramide, but not with S1P or SFA1P concentration. Allergen challenge increased plasma S1P concentration during EAR, but only in patients who developed both EAR and LAR. The magnitude of the increase determined during EAR correlated with the severity of subsequently developed LAR. Platelet and eosinophil counts were independent predictors of plasma S1P concentration. A significant increase in plasma SFA concentration in response to allergen challenge was seen only in patients who did not develop asthmatic response.ConclusionsAltered sphingolipid metabolism, with augmented synthesis of S1P and impaired de novo sphingolipid synthesis in response to allergen challenge, may participate in the development of asthma phenotype in HDM-APs.

Subcutaneous immunotherapy induces alterations in monocytes and dendritic cells homeostasis in allergic rhinitis patients

BackgroundSpecific subcutaneous immunotherapy (SCIT) can achieve long-term remission in patients with allergic rhinitis (AR) through complex and still unknown mechanisms. The aim of this study is to evaluate the effect of SCIT over CD16+ and CD16− monocytes, myeloid (mDCs) and plasmacytoid dendritic cells (pDCs) in patients with AR, comparatively to pharmacological standard treatment (non-SIT).MethodsThe relative frequency and absolute number of monocytes and DC subsets, the frequency of these cells producing TNFα after in vitro stimulation with Dermatophagoides pteronyssinus (Dpt) extract, and the expression levels of receptor-bound IgE or IgG were assessed by flow cytometry, in peripheral blood samples from 23 healthy individuals (HG) and 43 participants with AR mono-sensitized to Dpt; 10 with non-SIT treatment and 33 under SCIT, just before (SCIT-T0) and 4 h after administration (SCIT-T4). Moreover, IFNα mRNA expression was evaluated in purified pDCs, by qRT-PCR.ResultsAfter SCIT administration we observed a strong decrease of circulating pDCs, although accompanied by higher levels of IFNα mRNA expression, and an increase of circulating CD16+ monocytes. AR participants under SCIT exhibited a higher expression of receptor-bound IgE in all cell populations that expressed the high affinity receptor for IgE (FcεRI) and a higher frequency of CD16+ monocytes producing TNFα. Conversely, we observed a decrease in the frequency of mDCs producing TNFα in AR under SCIT, similar to the observed in the control group.ConclusionsSCIT seems to induce numeric, phenotypic, and functional changes in circulating monocytes and dendritic cells, contributing at least in part to the well described immunological alterations induced by this type of immunotherapy.

Synergistic effect of Dermatophagoides pteronyssinus allergen and Escherichia coli lipopolysaccharide on human blood cells.

PurposeHouse dust mites Dermatophagoides pteronyssinus are the main source of major inhalatory allergens inducing inflammatory response. Mite extract contain both allergenic proteins and lipopolysaccharides (LPS). The main allergenic protein, Der p 2, is a functional homolog of sMD-2, a protein providing blood cell response on LPS. Der p 2 may restore the response to LPS in absence of MD-2, but its interaction with LPS in whole blood is unknown. We studied the effect of Der p 2 on LPS-mediated activation of human whole blood cells.MethodsInteraction of Der p 2 and LPS was studied on eight healthy donors. The whole blood was incubated with extract of house dust mite Dermatophagoides pteronyssinus (DP-e), recombinant antigenic protein Der p 2 variant 5 (rDep 2), Escherichia coli lipopolysaccharide and their combination. Supernatants were collected for ELISA analysis of protein content. Activation degree was determined by change in concentration of TNF-α, IL-8, IL-1Ra cytokines and sMD-2 protein.Resultsextract of mite Dermatophagoides pteronyssinus (DP-e) possessed weak inherent activity and did not cause significant increase of cytokine production. Simultaneous activation of blood cells by LPS and DP-e led to considerable increase of pro-inflammatory cytokine production. We have shown the intrinsic inducing activity of Der p 2 allergen on sMD-2 protein and TNF-α cytokine expression.ConclusionsDer p 2 allergen enhances the response of human whole blood cells to external LPS by inducing additional expression of LPS-transporting protein sMD-2. The obtained data show an important role of LPS contamination of allegrens in the progress of allergic inflammatory response.

Low Potency of Indian Dust Mite Allergen Skin Prick Test Extracts Compared to FDA-Approved Extracts: A Double-Blinded Randomized Control Trial.

BackgroundSkin prick testing is the most important diagnostic tool to detect immunoglobulin E-mediated allergic diseases. With increase in the number of allergy tests performed in India, it is imperative to know the potency of indigenous extracts in comparison with U.S. Food and Drug Administration (USFDA)-approved extracts.MethodsA randomized comparison trial of Indian manufactured and USFDA-approved extracts of Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) was done at Christian Medical College & Hospital, Vellore, India from April 2014 to June 2015, to compare the skin test reactivity of indigenous allergen extracts of dust mites against validated allergen. Study enrollment included 197 patients with allergic disorders that showed sensitivity to dust mite during routine allergy skin testing. Study participants were tested with varying dilutions of DP and DF indigenous extracts along with USFDA-approved allergens in a blinded fashion. Results were recorded, and statistical significance was calculated using the Friedman rank sum test.ResultsUsing the Friedman rank sum test with a Tukey adjustment for multiple comparisons, we found that the extracts in each dilution were significantly different (P < .0001). The full strength indigenous extracts, B-DF (DF allergen standard extract from Bioproducts and Diagnostics, India) and C-DF (DF allergen extract from Creative Diagnostics, India) extracts, had mean wheal sizes of 7.69 (standard deviation [SD] 9.91) and 31.01(SD 51.04), respectively. The full strength S-DF (DF allergen extract from Jubilant Hollister Stier, Spokane, WA, USA) had a mean wheal size of 109.97 (SD 162.73), which was significantly higher (P < .0001) than both the indigenous extracts. For each of the dilutions, the S-DF mean wheal size was significantly greater than that of the corresponding B-DF and C-DF wheal sizes. The full strength indigenous C-DP (DP allergen extract from Creative Diagnostics, India) had mean wheal size of 39.37 (SD 51.74). The full strength standard S-DP (DP allergen extract from Jubilant Hollister Stier, Spokane, WA, USA) extract had a mean wheal size of 167.66 (SD 270.80), which was significantly higher (P < .0001) than the indigenous C-DP extract. Similar differences were seen across all dilutions.ConclusionThe indigenous extracts have significantly lower potency compared to USFDA-approved extracts; hence, there is an urgent need for policy makers to institute stringent criteria for standardization of antigens in India.

(S)-(+)-Decursin and its Analogues as Inhibitors of Cytokines Secretion Induced by DpE in THP-1/EoL-1 Cell Line

(S)-(+)-Decursin and its analogues were synthesized and evaluated for their inhibitory effects on monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and IL-6 secretion induced by Dermatophagoides pteronyssinus extract (DpE) in human acute monocytic leukemia (THP-1) cells and eosinophilic leukemia (EoL-1) cell lines. (S)-(+)- Decursin analogues with alkyl acryloyl groups, apart from (S)-(+)-decursin (1) and (S)-(+)-decursinol angelate (4a), did not influence the regulation of cytokine secretion. (S)-(+)-Decursin analogues (8d, 8f, 8g) with cinnamoyl groups carrying hydroxyl- and acetoxy-substitutes potently inhibited the secretion of MCP-1 and IL-6. The structure-activity relationship study revealed that increasing π-rich environment at the extensions from the eastern alcohol of (S)-(+)-decursin is a key element in building the pharmacophore for achieving the desired biological activity.

Safety of subcutaneous immunotherapy with tyrosine-adsorbed house dust mite extracts in patients with allergic disease

In spite of allergen-specific immunotherapy (SIT) multiple benefits, its use is restricted in some countries owing to concerns about severe adverse reactions. To evaluate systemic adverse reactions in patients with atopic dermatitis, allergic asthma, allergic rhinitis and allergic conjunctivitis who received subcutaneous immunotherapy with tyrosine-adsorbed Dermatophagoides and Glycyphagoides dust mites extracts. Retrospective study of the 2010-2015-period that included 773 patients diagnosed with IgE-mediated diseases, where the safety of allergen-specific immunotherapy was described according to the World Organization of Allergy subcutaneous immunotherapy-induced systemic reactions classification system. 79.7 % of patients had rhinitis, 54.9 % asthma, 34.5 % conjunctivitis and 16.4 % atopic dermatitis. Out of 12,546 tyrosine-adsorbed extract doses, 45 systemic reactions were recorded: 12 were grade 1 (30 %), 27 grade 2 (67.5 %) and 1 was grade 3 (2.5 %); the reaction rate was 0.35 per 100 administered injections, for an incidence rate of 5.8 %. No fatal reactions occurred. The frequency of systemic reactions with subcutaneous immunotherapy with Dermatophagoides farinae, Dermatophagoides pteronyssinus and Blomia tropicalis tyrosine-adsorbed extracts was similar to that reported with other extracts.

MONOMERIC ALLERGOID FROM HOUSE DUST MITE DERMATOPHAGOIDES PTERONYSSINUS: IMMUNOLOGICAL PROPERTIES

Background. The purpose of this study was to characterize the immunologic properties of the monomeric allergoid obtained from house dust mite Dermatophagoides pteronyssinus (D. pteronyssinus) extract. Methods. To obtain monomeric allergoid the extract from D. pteronyssinus (D1) was chemically modified by succinilation and then the degree of modification and total protein content were determined. Analysis of proteins in native D1 or modified sD1 was performed by SDS-PAGE. Determination of the major allergen Der p 1 in the native extract D1 was done by mass-spectrometry method. For measurement of tertiary structure alterations of protein molecules in D1 or recombinant Der p 1 (rDer p 1) after their modification by succinilation the circular dichroism (CD) method was applied. Allergenicity of sD1 was estimated by inhibition of specific IgE binding in ELISA and chemiluminescent analysis using Phadia ImmunoCAP as well as by detection of histamine released from leukocytes of whole blood of Der p sensitized patients after its incubation with D1 or sD1. To evaluate immunogenicity of D1 and sD1 BALB/c mice were i.p. immunized 4 times in 3 week interval with D1 or sD1 with or without adjuvant Al(OH),. The levels of anti-Der p IgE, IgG1 and IgG2a in pulled mice sera were determined by ELISA. Results. Succinilation of D1 extract led to «unfolding» of allergen molecules in modified sD1. The degree ofmodification was 98,9%. Results of SDS-PAGE demonstrated that native D1 and succinilated sD1 extract showed bands at the same molecular mass confirming that succinilation does not influence the size of modified molecule which thus maintains the monomeric nature of the sD1. Mass-spectrometry analysis revealed the presence of major allergen Der p 1 in the native D1 extract. CD method demonstrated the alteration of tertiary structure of succinilated recombinant rDer p 1 that may be extrapolated on the other allergenic proteins in the sD1 extract. Allergenicity of sD1 was significantly reduced in compare to D1 that was confirmed by inhibition of IgE-binding and histamine release from leukocytes of whole blood, Immunization of BALB/c mice with sD1 (with or without Al(OH)3) induced anti-Der p IgE-response which was substantially lower than that of D1 induced. At the same time anti-Der p IgG1 and IgG2a responses after immunization with sD1 (with or without Al(OH)3) were significantly higher than that ofD1 induced. Conclusion. Modification of the extract from house dust mite D. pteronyssinus by succinilation leads to formation of monomeric allergoid possesses low allergenicity and preserved (or even increased) immunogenicity that may a new approach for creation of allergy vaccines for safe and effective allergen-specific immunotherapy.

Suppression of Immunotherapy on Group 2 Innate Lymphoid Cells in Allergic Rhinitis.

Background:Group 2 innate lymphoid cells (ILC2s) are regarded as a novel population of lineage-negative cells that induce innate Type 2 responses by producing the critical Th2-type cytokines interleukin (IL)-5 and IL-13. ILC2s as key players in the development of allergic rhinitis (AR) have been proved, however, the effect of subcutaneous immunotherapy (SCIT) with dermatophagoides pteronyssinus extract (Der p-SCIT) on ILC2s in AR patients is not clear. This study aimed to investigate the response of ILC2s of peripheral blood in house dust mites (HDM)-sensitized Chinese patients with AR who received SCIT with Der P extract.Methods:Seven healthy controls without symptoms of AR who had negative reactions to any of the allergens from skin-prick testing, nine patients diagnosed with persistent AR according to the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines, and 24 AR patients who received Der p-SCIT for 1.0–3.5 years were recruited for the study. ILC2s in the peripheral blood were evaluated using flow cytometry. The severity of their symptoms of all participants was rated based on the Total 5 symptom score.Results:Among 40 participants, 9 AR patients were assigned to the untreated group, 24 AR patients receiving Der p-SCIT were assigned to the immunotherapy group, and 7 healthy controls without symptoms of AR were assigned to healthy control group. The mean Total 5 symptom score of immunotherapy group was significantly lower than that of untreated group (4.3 ± 1.4 vs. 10.1 ± 2.5, P < 0.001). Similarly, the levels of ILC2s in the peripheral blood of immunotherapy group were significantly reduced compared with that in untreated group (P < 0.001), but were not significantly different from healthy controls (P = 0.775). Further subgroup analysis based on the duration of SCIT therapy (1.0–2.0 years [SCIT1-2], 2.0–3.0 years [SCIT2-3], and 3.0–3.5 years [SCIT3-3.5]) showed that the percentage of ILC2s was not significantly different between SCIT1-2, SCIT2-3, and SCIT3-3.5 groups (SCIT1-2 vs. SCIT2-3: P = 0.268; SCIT1-2 vs. SCIT3-3.5: P = 0.635; and SCIT2-3 vs. SCIT3-3.5: P = 0.787).Conclusions:The present study highlighted the suppression of Der p-SCIT on ILC2s in HDM-AR patients. ILC2s identified in peripheral blood can be used as an effective biomarker for Der p-SCIT.

Secretoglobin Superfamily Protein SCGB3A2 Alleviates House Dust Mite-Induced Allergic Airway Inflammation in Mice

Background: Secretoglobin (SCGB) 3A2, a novel, lung-enriched, cytokine-like, secreted protein of small molecular weight, was demonstrated to exhibit various biological functions including anti-inflammatory, antifibrotic and growth-factor activities. Anti-inflammatory activity was uncovered using the ovalbumin-induced allergic airway inflammation model. However, further validation of this activity using knockout mice in a different allergic inflammation model is necessary in order to establish the antiallergic inflammatory role for this protein. Methods:Scgb3a2-null (Scgb3a2^-/-) mice were subjected to nasal inhalation of Dermatophagoides pteronyssinus extract for 5 days/week for 5 consecutive weeks; control mice received nasal inhalation of saline as a comparator. Airway inflammation was assessed by histological analysis, the number of inflammatory cells and various Th2-type cytokine levels in the lungs and bronchoalveolar lavage fluids by qRT-PCR and ELISA, respectively. Results: Exacerbated inflammation was found in the airway of Scgb3a2^-/- mice subjected to house dust mite (HDM)-induced allergic airway inflammation compared with saline-treated control groups. All the inflammation end points were increased in the Scgb3a2^-/- mice. The Ccr4 and Ccl17 mRNA levels were higher in HDM-treated lungs of Scgb3a2^-/- mice than wild-type mice or saline-treated Scgb3a2^-/- mice, whereas no changes were observed for Ccr3 and Ccl11 mRNA levels. Conclusions: These results demonstrate that SCGB3A2 has an anti-inflammatory activity in the HDM-induced allergic airway inflammation model, in which SCGB3A2 may modulate the CCR4-CCL17 pathway. SCGB3A2 may provide a useful tool to treat allergic airway inflammation, and further studies on the levels and function of SCGB3A2 in asthmatic patients are warranted.

The proportion of local allergic rhinitis to Dermatophagoides pteronyssinus in children.

Local allergic rhinitis (LAR) is diagnosed by the positive response to nasal allergen provocation test (NAPT) to aeroallergen and/or local synthesis of specific IgE (sIgE). This entity is found in half of the adults with non-allergic rhinitis (NAR). In children, very few data of the prevalence and characteristics of LAR were reported. Children 8-18years with NAR were recruited. A NAPT with Dermatophagoides pteronyssinus extract (NAPT-Dp) at 200, 600, and 2000AU/ml, respectively, at 15-min interval was performed. The immediate response was assessed using the clinical symptom score, peak nasal inspiratory flow (PNIF), and acoustic rhinometry (ARM). The nasal tryptase and sIgE-Dp were measured at baseline and 15min and 1h after a positive NAPT-Dp. Two allergic rhinitis (AR) patients were used as positive controls. Fifty-four NAR children (61.1% boys) with the mean±SD age of 11.1±2.1years were enrolled. The median duration of disease was 6.3years. The most frequent comorbidity was asthma (38.9%). Eighty-seven percent of patients had mild persistent severity. NAPT-Dp was positive in 2/54 (3.7%) of NAR children who had increased symptom score and decreased minimal cross-sectional area (MCA) on ARM as well as PNIF. However, there was no change in the nasal tryptase and sIgE-Dp. The control AR patients had positive NAPT-Dp and increased nasal tryptase at 15min without the change of sIgE-Dp. LAR is an uncommon condition in children. Further investigation in a large population of children with NAR is needed.

Are Dust Mite Allergens More Abundant or More Stable Than Other Dermatophagoides Pteronyssinus Proteins?

It is frequently reported that allergens are more stable and/or more abundant than other proteins. However, numeric comparisons on a genomic and proteomic scale are lacking. Dermatophagoides pteronyssinus (DP) expression levels were analyzed by RNAseq to assess the relative abundance of all transcripts. Thermodynamic stabilities of proteins in a DP extract were assessed using a mass spectrometry- and chemical denaturation-based strategy. Allergens are among the more highly expressed genes in DP. However allergens are not the most abundant, nor are the allergens a majority of the highly abundant genes. The thermodynamic stabilities of the 919 DP proteins assayed here were on average similar to those previously measured in other eukaryotic species. The 21 DP allergens identified trend toward greater stability than the remaining 898 proteins, but in general the allergens are not exceptionally stable; e.g. Der p 1’s stability is close to the mean. Der p 2 is greater than two standard deviations from the mean, however about 50 other non-allergens were also as stable as Der p 2. When considered together, high abundance and high stability increase the likelihood that a protein is an allergen, however it is not predictive. Allergens are among the more abundant and more stable proteins in DP. However, these features are not unique. In DP extract, commonly used for immunotherapy, the 898 non-allergenic proteins assayed had similar abundance and stability profiles to the 21 known allergens assayed. Abundance and stability do not completely account for the allergenic nature of some proteins.

Experimental mouse model of bronchial asthma induced by house dust mite Dermatophagoides pteronyssinus allergenic extract

The purpose of this study was to develop a mouse model of asthma (MMA) using house dust mite Dermatophagoides pteronyssinus (Der p) extract. Methods. BALB/c mice were i.p. immunized with different doses of Der p lyophilized extract three times in three week interval in the mixture with Al(OH)3. 8 weeks after the final immunization mice were challenged with Der p during five consecutive days by intranasal applications (INA) or aerosol administration (AA). All mice were divided into 5 experimental groups: group 1 was immunized with 50 ^g/mouse of Der p (in protein equivalent) in the mixture with 2 mg/mouse Al(OH)3 and challenged by INA; group 2 was immunized in the same way and challenged by AA; group 3 was immunized with 100 ^g/mouse Der p in the mixture with 2 mg/mouse of Al(OH)3 and challenged by INA; group was immunized in the same way and challenged by AA; group 5 (negative control) was immunized and challenged with saline. 24 hours after the last challenge airway hyperresponsiveness (AHR) to different concentrations of metha-choline was evaluated in all groups by whole-body plethysmography. 48 hours after the last challenge in all groups blood was collected for differential cell count, brochoalveolar lavage fluid (BALF) was sampled for the determination of inflammatory cells and lungs were removed for histological analysis. Histopathological changes in lungs (allergic inflammation) were graded according to semi-quantitative scoring system. Anti-Der p IgE, IgG1 and IgG2a antibodies in individual sera samples were detected by ELISA seven days after the last immunization and 48 hours after the challenge. Results. The levels of anti-Der p IgE antibodies in groups 1-4 before as well as after the challenge were substantially higher than that of in the group 5 (negative control). The highest level of serum Der p-specific IgE antibodies was observed in the group 2. The levels of anti-Der p IgG1 and IgG2a antibodies in the groups 1-4 during all periods of observation were higher than that of group 5 (negative control). At the same time the maximal levels of anti-Der p IgG1 and IgG2a antibodies were observed in group 3 both after immunization and after challenge. The maximum of AHR was observed in the groups 1 and 3 challenged by INA. Analysis of cell composition in BALF demonstrated significant elevated number of eosinophils in group 3 in comparison with group 5 (negative control) and other experimental groups. Regarding peripheral blood leukocyte count we observed decreasing of band neutrophils in group 4 and increasing of segmented neutrophils in groups 1 and 3 in compare to group 5 (negative control). In group 1 we found statistically significant decreasing of lymphocytes and increasing of eosinophils in compare to negative control group 5. Histological picture of general allergic inflammation in lungs as well as peribronchial and perivascular infiltration with inflammatory cells were the most noticeable (according to score system) in group 3 in comparison with negative control group and other experimental groups. Conclusion. Data obtained indicate that immunization (sensitization) of mice with Der p in a dose 100 μg/mouse together with Al(OH)3 and challenge with Der p by mean of intranasal applications is a suitable approach for modeling of mouse allergic asthma.

House dust mite allergen suppresses neutrophil apoptosis by cytokine release via PAR2 in normal and allergic lymphocytes.

House dust mite (HDM) is an essential allergen in allergic diseases such as allergic rhinitis and asthma. The pathogenic mechanism of allergy is associated with cytokine release of lymphocytes and constitutive apoptosis of neutrophils. In this study, we examined whether HDM induces cytokine release of lymphocytes and whether the secretion of cytokines is involved in modulation of neutrophil apoptosis. In normal and allergic subjects, extract of Dermatophagoides pteronyssinus (DP) increased IL-6, IL-8, MCP-1, and GM-CSF secretion in a time-dependent manner. This secretion was suppressed by PAR2i, an inhibitor of PAR2, in a dose-dependent manner, as well as by LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, and BAY-11-7085, and an inhibitor of NF-κB. DP induced ERK and NF-κB activation in a time-dependent manner. ERK activation was suppressed by PAR2i, LY294002, and AKTi, and NF-κB activation was blocked by PAR2i, LY294002, AKTi, and PD98059. Supernatants collected from normal and allergic neutrophils after DP treatment inhibited the apoptosis of normal and allergic neutrophils through suppression of caspase 9 and caspase 3 cleavage. DP inhibited neutrophil apoptosis in coculture of normal neutrophils with normal lymphocytes, similar to the anti-apoptotic effects of DP on neutrophils alone. DP more strongly inhibited apoptosis of allergic neutrophils cocultured with allergic lymphocytes than allergic neutrophils without lymphocytes. In summary, DP induces the release of cytokines through the PAR2/PI3K/Akt/ERK/NF-κB pathway, which has anti-apoptotic effects on neutrophils of normal and allergic subjects. These results will facilitate elucidation of the pathogenic mechanism of allergic diseases.

Cloning and characterization of tropomyosin from the mite Chortoglyphus arcuatus.

Tropomyosin is a pan-allergen that shares a high homology among species. It is involved in cross-reactivity among mites, crustaceans, mollusks and insects. The objectives were to express and purify recombinant tropomyosin from the storage mite Chortoglyphus arcuatus, and to investigate the homology and cross-reactivity with tropomyosin from other invertebrates. Recombinant C. arcuatus tropomyosin (r-Cho a 10) was selected from a library by screening with a pool of patient sera. r-Cho a 10 (UniProt: H2DFL1) was sequenced, expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Polyclonal anti-tropomyosin antibodies were produced in mice. IgE recognition of r-Cho a 10 was checked by immunoblot. Immunoblot inhibition assays were used to identify the native tropomyosin in the complete extract from C. arcuatus and study cross-reactivity between r-Cho a 10 and Der p 10. Identification of tropomyosin in other allergenic sources was performed by immunoblot. r-Cho a 10 showed a high homology (54-96%) with other tropomyosins from different allergenic sources. IgE recognition was observed using a pool of sera from sensitized individuals. Tropomyosins from different extracts were identified not only in the whole C. arcuatus extract but also in Dermatophagoides pteronyssinus, shrimp, mussel, cockroach and Anisakis extracts with polyclonal α-Cho a 10. r-Cho a 10 completely inhibited the recognition of Der p 10. Recombinant C. arcuatus tropomyosin maintained its capacity to recognize IgE. r-Cho a 10 was used to prove cross-reactivity among tropomyosins from other invertebrate species, including mites. This is the first C. arcuatus allergen included in the WHO/IUIS (World Health Organization/International Union of Immunological Societies) Allergen Nomenclature database.

Evaluation of the Role of IgE Responses to Der p 1 and Der p 2 in Chinese House Dust Mite-Allergic Patients

Background: The role of specific IgE (sIgE) against Der p 1 and Der p 2 in Chinese patients with house dust mite (HDM) allergy has not yet been well investigated. Methods: One hundred patients were enrolled, based on sensitization and doctor-diagnosed allergy to HDM. Questionnaires were administered to document demographic and clinical characteristics. Serum IgE reactivity to Dermatophagoides pteronyssinus (Dp) extract, Der p 1, Der p 2 and Der p 10 was measured by ImmunoCAP. Results: Almost all patients were sensitized to Der p 1 (95%) and Der p 2 (93%), with both allergens together being largely responsible for the total anti-HDM IgE response. No evidence for a significant role of Der p 10 was found. Overall, IgE responses to HDM and its 2 major allergens were higher in children than in adults in this cross-sectional study. With increasing age, IgE responses to Der p 2 become more important. A positive correlation was observed between the reaction of sIgE against Dp, Der p 1 and Der p 2 and the number of organs (including the eyes, nose, lungs and skin) that were affected in patients. Conclusions: In China, Der p 1 and Der p 2 are the dominant allergens in patients with HDM allergy. The relative importance of Der p 1 and Der p 2 changes with age, in favor of Der p 2. Overall, sIgE titers were positively associated with the number of organs affected.

Anti-apoptotic Effects of House Dust Mite, S100A8 and S100A9 on Spontaneous Apoptosis of Neutrophils in Coculture with Immune Cells and in the Presence of T Helper Cytokines

House dust mite (HDM) as a major allergen and damage-associated molecular pattern (DAMP) such as S100A8 and S100A9 trigger the pathogenesis and severity of allergic disease such as asthma. Regulation of neutrophil apoptosis is an important immune response and its dysregulation is involved in pathogenesis of allergic diseases. In this study, we examined the effects of HDM, S100A8 and S100A9 on spontaneous apoptosis of normal neutrophils. We considered the importance of the difference between in vitro and in vivo results and developed a new in vitro system consisting of a combination of immune cells and T helper (Th) cytokines. Extract of Dermatophagoides pteronyssinus (DP), S100A8, and S100A9 inhibited neutrophil apoptosis in culture of neutrophils alone without other leukocytes. DP and S100A8 more strongly suppressed neutrophil apoptosis in combinations of neutrophils, eosinophils, lymphocytes or monocytes than in a culture of neutrophils alone. Anti-apoptotic effect of S100A9 in the mixture of immune cells was similar to that in neutrophils. DP, S100A8, and S100A9 blocked neutrophil apoptosis, regardless of pretreatment with a T helper (Th) 1 cytokine (IFN-γ), Th2 cytokines (IL-4 and IL-10), a Th9 cytokine (IL-9), a Th17 cytokine (IL-17), a Treg-producing cytokine (TGF-β). These findings may enable elucidation of allergy pathogenesis due to HDM and DAMP.

Investigation of the positivity profile for the skin prick test in children infected with parasites in the metropolitan region of Pernambuco, Northeast of Brazil

Methods Children between 2 and 14 year old were submitted to a parasitological survey using the Hoffman, Pons e Janer (3 blades) method and the Kato-Katz (2 blades) method. The ISAAC questionnaire was applied to collect informations related to asthma. The Skin Prick Test was applied using extracts of Dermatophagoides pteronyssinus (DP), Blomia tropicalis (BT), Blatella germânica (BG), Periplaneta americana (PA), fungus and cat epithelium (EPC).

The IgE response to Ascaris molecular components is associated with clinical indicators of asthma severity

Background Asthma is a common chronic disease worldwide and Ascaris lumbricoides infection (ascariasis) is frequent in tropical regions. However, the effect of ascariasis on asthma severity has not been sufficiently explored. We sought to evaluate the influence of the IgE immune response to Ascaris extract and purified house dust mites (HDM) and Ascaris allergens on indicators of asthma severity in patients living in the tropics.Methods Asthmatic patients from Cartagena, Colombia were recruited. Clinical assessment included questionnaires, physical examination, allergy skin tests, spirometry, parasite stool examination and IgE antibody measurements. Asthma was diagnosed by a physician according to validated criteria. Indicators of severity were occurrence of severe dyspnea episodes, night awakenings events, > 4 emergency room (ER) visits and hospitalizations during the last year. Specific IgE to Der p 2, Ascaris spp., Blomia tropicalis and Dermatophagoides pteronyssinus extracts was determined by ImmunoCap. IgE to tropomyosins (Asc l 3, Blo t 10 and Der p 10), Blo t 5 and Asc s 1 was detected by ELISA. Logistic regression analyses were used to explore the relationships between sensitization and indicators of asthma severity.Results After adjustment for HDM sensitization, Ascaris sensitization remained associated with severe dyspnea (aOR: 1.90, 95% CI: 1.08 - 3.34, p = 0.03) and > 4 ER visits (aOR: 2.23, 95% CI: 1.15 - 4.30, p = 0.02). We also found that sensitization to the species specific markers Blo t 5 and Asc s 1, as well as the cross-reactive tropomyosins of D. pteronyssinus and Ascaris were associated with > 4 ER visits. Der p 2 sensitization was associated with bronchodilator responsiveness (aOR: 2.24: 1.25-4.02, p = 0.01). Remarkably, significantly higher IgE levels to HDM species specific allergens were found in Ascaris sensitized patients.Conclusions In this tropical population, IgE sensitization to Ascaris and the cross-reactive tropomyosins was frequent and associated with clinical indicators of asthma severity. The significant relationship between sensitization to the nematode-specific marker Asc s 1 and ER attendance supports these findings. Moreover, ascariasis increases the human IgE responses to HDM specific allergens.