Tyrosine nitration enhances the allergenic potential of house dust mite allergen Der p 2.

Nitration of allergenic proteins caused by atmospheric pollutants O3 and NO2 may enhance their allergenic potential. In the study, the influence of nitration was investigated on the allergenicity of Der p 2, which is a main allergen from house dust mites and plays an important role in allergenic rhinitis and asthma. The results reveal that nitrated Der p 2 enhanced the IgE-binding capacity, upregulated the mRNA expression and release of IL-6 and IL-8 from bronchial epithelial cells, and induced higher levels of specific-IgE, TH2 cytokines and white blood cells in mice. Besides, nitrated Der p 2 caused more severe oxidative stress and allergenic symptoms in mice. It is concluded that nitration enhanced the allergenicity of Der p 2 through not only directly inducing higher amount of specific-IgE and stronger responses of TH2 cytokines, but also indirectly aggravating allergic symptoms by oxidative stress and adjuvant-like activation airway epithelial cells. The study suggests that the contribution of nitration to the promotion in allergenicity should not be ignored when precisely assessing the risk of house dust mite allergens in real environment.

Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas).

BACKGROUND: House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. OBJECTIVES: To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. METHODS: Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. RESULTS: The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). A positive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CONCLUSIONS: CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.

Sialic acid-modified der p 2 allergen exerts immunomodulatory effects on human PBMCs.

Background: House dust mite extract-based allergen immunotherapy (AIT) to treat house dust mite allergy is substantially effective but still presents some safety and efficacy concerns that warrant improvement. Several major allergen-based approaches to increase safety and efficacy of AIT have been proposed. One of them is the use of the group 2 allergen, Der p 2. Objective: We sought to investigate the immunomodulatory effects of sialic acid-modified major allergen recombinant Der p 2 (sia-rDer p 2) on PBMCs from healthy volunteers. Methods: We activated PBMCs with anti-CD3/CD28 antibodies and incubated them at 37°C for 6 days in the presence or absence of either native rDer p 2 or α2-3 sialic acid-modified rDer p 2 (sia-rDer p 2). We assessed the changes in CD4+ T-cell activation and proliferation by flow cytometry and changes in T-lymphocyte cytokine production in cell culture supernatant by ELISA. Results: We observed that PBMCs treated with sia-rDer p 2 presented with a markedly decreased expression of CD69 and an increased abundance of LAG-3+ lymphocytes compared with cells treated with rDer p 2. Moreover, PBMCs treated with sia-rDer p 2 showed a reduced production of IL-4, IL-13, and IL-5 and displayed a higher IL-10/IL-5 ratio compared with rDer p 2-treated PBMCs. Conclusions: We demonstrate that sia-rDer p 2 might be a safer option than native rDer p 2 for Der p 2-specific AIT. This is most relevant in the early phase of AIT that is often characterized by heightened TH2 responses, because sia-rDer p 2 does not enhance the production of TH2 cytokines.

Corrigendum: Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

[This corrects the article DOI: 10.3389/fimmu.2023.1155613.].

Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 cytokine-driven skin inflammation and epithelial barrier dysfunction. The latter is believed to allow the increased penetration of chemicals, toxins, and allergens into the skin. House dust mite allergens, particularly Der p 2, are important triggers in sensitized individuals with AD; the precise actions of these allergens in epithelial biology remain, however, incompletely understood. In this study, we compared the effects of the protein allergen Der p 2 and a mix of non-IgE-reactive Der p 2 peptides on skin cells using patch tests in AD patients and healthy participants. We then analyzed mRNA expression profiles of keratinocytes by single-cell RNA-sequencing. We report that existing barrier deficiencies in the non-lesional skin of AD patients allow deep penetration of Der p 2 and its peptides, leading to local microinflammation. Der p 2 protein specifically upregulated genes involved in the innate immune system, stress, and danger signals in suprabasal KC. Der p 2 peptides further downregulated skin barrier genes, in particular the expression of genes involved in cell-matrix and cell-cell adhesion. Peptides also induced genes involved in hyperproliferation and caused disturbances in keratinocyte differentiation. Furthermore, inflammasome-relevant genes and IL18 were overexpressed, while KRT1 was downregulated. Our data suggest that Der p 2 peptides contribute to AD initiation and exacerbation by augmenting hallmark features of AD, such as skin inflammation, barrier disruption, and hyperplasia of keratinocytes.

Co-Activation of Human Whole Blood Cells with Lipopolysaccharides and an Allergen.

The investigation of common inflammation mechanisms caused by exogenic compounds of microbial origin and allergens is one of the most important tasks in current biomedical science. The main manifestations of immune cell activation caused by pro-inflammatory agents are changes in receptor quantity on the surface of immune cells and the production of cytokines and chemokines by blood cells. The levels of expression of TLR4, CD14, and CD11b in the monocytes and neutrophils of human whole blood in response to LPS E. coli, Der p 2 allergen, or their combination reflect different functional activities in these cells, while the composition and amount of produced cytokines reflect the biological activity of the studied agonists. The activity of Der p 2 allergen in ex vivo experiments on whole blood samples is significantly lower compared with its activity in vitro in isolated PBMC cells, which should be taken into account when transferring the results obtained for isolated cells to whole blood cells. LPS R. capsulatus PG significantly decreases the synthesis of MyD88-dependent NF-κB-regulated cytokines activated by LPS E. coli, Der p 2, or their combination. This indirectly indicates the general mechanisms of cell activation caused by these structures and the unified mechanism of the protective action of LPS R. capsulatus PG against both endotoxin and a combination of endotoxin and the allergen.

Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens.

Background: House dust mite (HDM) is a major cause of respiratory allergic diseases. Dendritic cells (DCs) play a central role in orchestrating adaptive allergic immune responses. However, it remains unclear how DCs become activated by HDM. Biochemical functions of the major HDM allergens Der p 1 (cysteine protease) and Der p 2 (MD2-mimick) have been implicated to contribute to DC activation. Methods: We investigated the immune activating potential of HDM extract and its major allergens Der p 1 and Der p 2 using monocyte-derived DCs (moDCs). Maturation and activation markers were monitored by flow cytometry and cytokine production by ELISA. Allergen depletion and proteinase K digestion were used to investigate the involvement of proteins, and in particular of the major allergens. Inhibitors of spleen tyrosine kinase (Syk), Toll-like receptor 4 (TLR4) and of C-type lectin receptors (CLRs) were used to identify the involved receptors. The contribution of endotoxins in moDC activation was assessed by their removal from HDM extract. Results: HDM extract induced DC maturation and cytokine responses in contrast to the natural purified major allergens Der p 1 and Der p 2. Proteinase K digestion and removal of Der p 1 or Der p 2 did not alter the immune stimulatory capacity of HDM extract. Antibodies against the CLRs Dectin-1, Dectin-2, and DC-SIGN did not affect cytokine responses. In contrast, Syk inhibition partially reduced IL-6, IL-12 and completely blocked IL-10. Blocking TLR4 signaling reduced the HDM-induced IL-10 and IL-12p70 induction, but not IL-6, while endotoxin removal potently abolished the induced cytokine response. Conclusion: Our data strongly suggest that HDM-induced DC activation is neither dependent on Der p 1 nor Der p 2, but depend on Syk and TLR4 activation, which might suggest a crosstalk between Syk and TLR4 pathways. Our data highlight that endotoxins play a potent role in immune responses targeting HDM.

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Background: Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Methods: Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (ß-hexosaminidase) release. Results: One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. Conclusion: The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

Immunomodulatory effects of modified Liu-Wei-Di-Huang-Wan Traditional Chinese medicine on allergic asthmatic mice.

BACKGROUND: Allergic asthma occurs worldwide and is particularly prevalent in westernized countries characterized by chronic airway inflammation resulting in airway hyperresponsiveness. The house dust mites (HDM) including Dermatophagoides pteronyssinus are major sources of sensitization and triggering allergic symptoms in asthmatic patients. The Der p 2 is a major allergen and the predominant source of causative respiratory disorders which induce airway inflammation and bronchial constriction in mite-allergic patients. Few studies evaluate the ameliorating effects of modified Liu-Wei-Di-Huang-Wan (modified LWDHW) on allergic asthma. METHODS: This study aimed to investigate the immunological mechanisms of modified LWDHW on the reductions of airway inflammation, signal transduction, inflammatory cytokine production, Th2 cell proliferation, and bronchial obstruction in Der p 2-induced asthmatic mice. RESULTS: At least ten active ingredients were contained in the formula of modified LWDHW- 1217A and 1217B. Results showed that the immunoglobulin generations (Der p 2 specific- IgE and IgG1), inflammatory cytokine productions (IL-5 and IL-13) in the Sera and BALF could be down-regulated, and the Th1-cytokine productions (IL-12 and IFN-γ) be increased after immunotherapy with modified LWDHW of 1217A or 1217B. The inflammatory cell infiltrations (macrophages, eosinophils, and neutrophils) in the airway and the expressions of TH2-related genes (IL-4, IL-5, and IL-13), TH2-related transcription factor (GATA-3), and neutrophil chemotactic chemokine (IL-8) in the lung tissue of asthmatic mice were significantly decreased after the immunotherapy. The Th1/Th2 polarization had been identified that the IL-4+/CD4+ T cells were downregulated and IFN-γ+/CD4+ T cells were increased. The airway hyperresponsiveness to methacholine inhalation of Penh values was significantly decreased in the treated groups. There were significant improvements in the bronchus histopathology after immunotherapy with 1217A or 1217B which were evaluated by tracheal thickness, inflammatory cell count, and tracheal rupture of mouse lung. CONCLUSION: It revealed that 1217A or 1217B could regulate the immune responses and improve pulmonary function. Data suggests that modified LWDHW of 1217A or 1217B have the potential for use as a therapeutic intervention for the treatment of mite allergen Der p 2-induced allergic asthma.

Participation of MAPK and PI3K in Regulation of Cytokine Secretion by Peripheral Blood Monocular Cells in Response to Escherichia coli LPS and rDer p 2 Combination.

Search for the effective approaches to treat acute inflammation caused by combination of allergens and infectious agents is an important task for public health worldwide. House dust mites Dermatophagoides pteronyssinus are the source of allergens of the Der p groups and of microbial compounds, in particular, lipopolysaccharides (LPS). LPS and Der p 2 induce secretion of pro-inflammatory cytokines via activation of kinases p38 MAPK, MEK1/2, and PI3K. Participation of these kinases in the regulation of cells response to combined exposure to LPS and Der p 2 has not been sufficiently studied. We studied the effects of kinases (p38 MAPK, MEK1/2, and PI3K) inhibition on secretion of cytokines (TNF, IL-8, and IL-6) by peripheral blood mononuclear cells (PBMC) of healthy volunteers in response to E. coli LPS and rDer p 2. Contribution of kinases to the regulation of cell response to different agents (rDer p 2 and/or LPS) was revealed. It was found that p38 MAPK plays a key role in the regulation of secretion TNF by PBMC in response to the combination of LPS and rDer p 2. MEK1/2-dependent signaling is the main pathway for the synthesis of TNF and IL-8 in response to LPS and rDer p 2. PI3K-dependent signaling negatively regulates TNF production during rDer p 2-induced cell activation, but is not involved in the response to the combination of LPS and rDer p 2. PI3K-dependent signaling in the regulation of PBMC cytokine synthesis is most pronounced in response to their activation by rDer p 2. Understanding the mechanisms of immune cell responses to combinations of inflammatory agents could facilitate the search for new intracellular targets for anti-inflammatory therapy.

Effect of combination treatment with Lactobacillus rhamnosus and corticosteroid in reducing airway inflammation in a mouse asthma model.

BACKGROUND: Asthma is a complex multifactorial chronic airway inflammatory disease with diverse phenotypes and levels of severity and is associated with significant health and economic burden. In a certain population of asthma patients, the symptoms cannot be well controlled with steroid. There has been long standing interest in the use of probiotics for treating allergic diseases. The purpose of this study is to investigate whether the combination of Lactobacillus rhamnosus GG (LGG) with prednisolone could reduce the dosage of glucocorticoid in controlling airway inflammation in a murine model for allergic asthma. MATERIAL AND METHODS: We used Der p 2-sensitized asthma model in female BALB/c mice. The animals were treated with 75 µl or 50 µl oral prednisolone or combination treatment of these two doses of oral prednisolone with LGG. Airway hyperresponsiveness, serum specific IgE/IgG1/IgG2a, infiltrating inflammatory cells in lung and cytokines were assessed. RESULTS: Compared to 75 µl prednisolone, a lower dose of prednisolone with 50 µl was less satisfactory in suppressing airway hyperresponsives, serum IgE and IgG1, Th2 cytokines and inflammatory cytokines such as IL-6, IL-8 and IL-17 as well as infiltrating inflammatory cells. However, combination of 50 µl prednisolone and LGG decreased airway resistance and serum IgE and IgG1, inhibited the production of IL-4, IL-5, IL-6, IL-8, IL-13 and IL-17, upregulated serum IgG2a and enhanced Th1 immune response. CONCLUSIONS: LGG may reduce the dosage of prednisolone and thus may be beneficial in the treatment of asthma.

Assessment of TSLP, IL 25 and IL 33 in patients with shrimp allergy.

BACKGROUND: Shrimp allergy is a growing problem among the European population. TSLP, IL-25 and IL-33 are involved in the pathophysiology of allergic diseases, including asthma and atopic dermatitis, as they activate the Th2-dependent immune response. METHODS: Thirty-seven patients (18 male and 19 female) with a positive history of symptoms associated with shrimp consumption were selected. All patients had blood samples taken to assess the concentration of allergen-specific IgE (sIgE) to house dust mites (HDM) and shrimp (Singleplex, quantitative method with cut off value > 0,35 kAU/L) as well as the level of allergen components using the ImmunoCap ISAC method (Microarray test, semi-quantitative with cut off value > 0,3 ISU-E). The concentrations of TSLP, IL-25 and IL-33 in the patients' blood serum was assessed using the ELISA method (Cusabio). Twenty patients with negative allergy history of allergic disease tests were included in the control group. RESULTS: Among the 37 shrimp-allergic patients, ImmunoCap ISAC was identified the presence of sIgE to the available shrimp allergen components in only 14 cases (37.8%). TSLP and IL25 levels were significantly higher in the study group. No statistically significant correlation was found between the concentration of analyzed alarmins and the concentration of sIgE level to shrimp or HDM between the study and control groups. No statistically significant correlation was found between poly-sensitization occurring in patients and levels of TSLP, IL-25 and IL-33 . CONCLUSION: In shrimp-allergic patients, the concentrations of TSLP and IL-25 were significantly higher than in the control group (1.33 vs. 0.49 and 157 vs. 39.36, respectively). There was no correlation between the concentrations of TSLP, IL-25 and IL-33 and the concentration of sIgE in the patients or the number of allergen components that the patients were sensitized to. TRIAL REGISTRATION: Bioethics Committee 147/2015, 11.03.2015.

Sensitization to major Dermatophagoides pteronyssinus allergens in house dust mite allergic patients from North Eastern Poland developing rhinitis or asthma.

PURPOSE: Recognition of individual allergens by IgE is crucial for triggering symptoms in allergic rhinitis (AR) or asthmatic (AA) patients. House dust mite (HDM) allergy is frequent around the world, the sensitization profile to individual HDM allergens varies in individual HDM-allergic patients (APs). The aim of this study was to evaluate the pattern of IgE sensitization to three major Dermatophagoides pteronyssinus (Dp) allergens among patients from North Eastern Poland suffering from HDM-AR and/or AA. PATIENTS AND METHODS: The study was performed on 323 HDM-AR and/or AA patients and 106 controls (CG) including 30 healthy non-atopic subjects, 32 AR patients not sensitized to Dp and 44 non-atopic asthmatics. IgE levels to natural (n)Der p 1, nDer p 2, recombinant (r)Der p 2.0101 and rDer p 23 allergens were measured by ELISA. RESULTS: The majority of HDM-APs were sensitized to nDer p 1 (72.1%), nDer p 2 (81.7%), rDer p 2.0101 (78.3%) and rDer p 23 (70.9%). The frequency of positive results to individual allergens depended on clinical manifestations and the level of IgE to the whole Dp extract. In HDM-AA patients, reactivity to nDer p 1 and rDer p 23 was detected more frequently than in HDM-AR patients. The whole Dp extract completely inhibited IgE binding to nDer p 1 and nDer p 2 but only partially to rDer p 23. CONCLUSIONS: HDM-APs from North-Eastern Poland display sensitization profile to major allergens which is similarly observed in western Europe. HDM-based diagnostic and therapeutic products should include all major allergens.

Molecular Diagnosis in House Dust Mite-Allergic Patients Suggests That Der p 23 Is Clinically Relevant in Asthmatic Children.

BACKGROUND: Patterns of sensitization to house dust mites depend on geographic area and are important in clinical practice. However, the role of molecular diagnosis is not currently defined. We sought to characterize a pediatric population by focusing on sensitization to different mite species and major mite components in order to assess the clinical relevance of sensitization to allergenic components in our practice. METHODS: Consecutive children with respiratory allergy sensitized to house dust mites (determined by skin prick test [SPT]) were recruited. We determined specific IgE to nDer p 1, rDer p 2, and rDer p 23 using ImmunoCAP and sIgE using ImmunoCAP-ISAC microarray. Patients were followed up for 3 years. RESULTS: A total of 276 children were recruited. The frequency of sensitization was 86.6% for nDer p 1, 79.3% for rDer p 2, and 75.8% for rDer p 23. Lepidoglyphus species was the most common storage mite detected by SPT. Twenty-six patients (9.4%) were not sensitized to Der p 1 or Der p 2. It is noteworthy that IgE binding to Der p 23 was positive in 14 (53.8%). Asthmatic patients, especially those with a persistent moderate-severe phenotype, more frequently recognized the 3 major allergens. CONCLUSIONS: Most patients with mite allergy were sensitized to the major allergens Der p 1, Der p 2, and Der p 23. Of the allergens evaluated, 5% were sensitized to Der p 23 but not to Der p 1 or Der p 2. Sensitization to Der p 23 should be considered in the diagnosis and treatment of mite allergy, especially in patients with moderate-severe asthma, because it may worsen the clinical phenotype.

CRM197-Coupled Der p 2 Peptides Suppress Allergic Airway Inflammation in a Der p 2-Induced Asthma Mouse Model.

Allergic diseases affect more than 25% of the global population. Der p 2 is the major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus. Allergen-specific immunotherapy is the only treatment to change the course of allergic diseases. In this study, two synthesized Der p 2 peptides coupled to cross-reacting material 197 (CRM197) showed reduced IgE reactivity and allergenic activity. CRM197-coupled Der p 2 peptides induced rDer p 2-specific IgG1 antibodies in mice, which could inhibit HDM-allergic patients' IgE binding to rDer p 2. The immunity effects of CRM197-coupled Der p 2 peptides were studied in an rDer p 2-induced asthma mouse model. CRM197-coupled Der p 2 peptides can suppress asthmatic airway inflammation in this model. Analysis of IL-4, IL-5, and IFN-γ levels in bronchoalveolar lavage fluid revealed that the suppression was associated with a shift from a Th2 to a Th1 response. Thus, CRM197-bound Der p 2 peptides exhibited less allergenic activity than the rDer p 2 allergen, which preserved immunogenicity and may be candidates for mite allergy vaccines.

Monocyte-Derived Dendritic Cells as Antigen-Presenting Cells in T-Cell Proliferation and Cytokine Production.

Dendritic cells (DC) are the most potent antigen-presenting cells that link innate with adaptive immunity. They circulate the body and sample the microenvironments for maintaining homeostasis and for mounting T-cell responses against invading pathogens, foreign antigens, and aberrant self-proteins. In humans, DC derived from blood monocytes (MDC) by cytokine treatment provide the most abundant and versatile source for studying DC and T-cell biology, and for use as adjuvants in cancer therapy. In asthma patients, T-cell functions are studied by using autologous MDC as accessory cells for allergen presentation. The method for isolating T cells and monocytes from peripheral blood mononuclear cells (PBMC) and the stimulation of T cells to proliferate and produce cytokines by MDC are outlined in this chapter. The method can be applied to the functional studies of T cells and DC in other diseases.

Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen.

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

The Dermatophagoides pteronyssinus Molecular Sensitization Profile of Allergic Rhinitis Patients in Central China.

Background House dust mites (HDMs) are the major aeroallergens in patients with rhinitis and/or asthma in China. However, the molecular sensitization of HDM is varied in different regions. Objective To investigate the Dermatophagoides pteronyssinus ( Der p) molecular sensitization profile of allergic rhinitis (AR) patients in Central China. Methods AR patients with positive skin prick tests to Der p were enrolled in our study. Specific immunoglobulin E (sIgE) for Der p, Dermatophagoides farinae ( Der f), Acarus siro ( Aca s), Blomia tropicalis ( Blo t), and Der p components Der p1, Der p2, and Der p10 were measured in all patients. Results A total of 130 patients were included. The overall prevalence of positive serum sIgE was 99.2% for Der p, 98.5% for Der f, 81.5% for Aca s, 83.1% for Blo t, 71.5% for Der p1, 64.6% for Der p2, and 11.5% for Der p10. HDM-AR patients with asthma displayed significantly higher concentrations of sIgE to Der p, Der f, Der p1, and Der p2 than did those without asthma ( P < .001). The prevalence of asthma in HDM-AR patients was higher among the patients sensitized to both Der p1 and Der p2 (62.8%) than those sensitized to only 1 allergen ( Der p1 26.7% or Der p2 16.7%; P < .05) or nonsensitized to Der p1 and Der p2 (19.4%; P < .001). Conclusion Der p has high cross-reactivity with other mite species. Der p1 and Der p2 are the major components to induce Der p sensitization among AR patients in Central China. Sensitization to both Der p1 and Der p2 may be a risk factor for developing asthma in HDM-AR patients.