Der P1 Research Articles

Enhanced Expression of IL-18 and IL-18BP in Plasma of Patients with Eczema: Altered Expression of IL-18BP and IL-18 Receptor on Mast Cells.

IL-18 has been found to be associated with eczema. However, little is known of the role of IL-18 binding protein (BP) and IL-18 receptor (R) in eczema. We therefore investigated the expression of IL-18, IL-18BP, and IL-18R on mast cells by using flow cytometry analysis and mouse eczema model. The results showed that plasma free IL-18 and free IL-18BP levels in eczema patients were higher than those in healthy controls. IL-18 provoked up to 3.1-fold increase in skin mast cells. IL-18 induced also an increase in IL-18BP+ mast cells, but a reduction of IL-18R+ mast cells in mouse eczema skin. It was found that house dust mite allergen Der p1 and egg allergen OVA induced upregulation of the expression of IL-18, IL-18BP, and IL-18R mRNAs in HMC-1 cells following 2 and 16 h incubation. In conclusion, correlation of IL-18 and IL-18BP in eczema plasma suggests an important balance between IL-18 and IL-18BP in eczema. The decrease in molar concentration ratio of plasma IL-18BP/IL-18 and allergen-induced upregulated expression of IL-18 and IL-18R in skin mast cells of the patients with eczema suggests that anti-IL-18 including IL-18BP therapy may be useful for the treatment of eczema.

A specific immune therapeutic effect of Der p2 T cell epitope vaccine on asthma mice

To investigate the specific immune therapeutic effect of the T cell fusion peptide vaccine from group II allergens from Dermatophagoides pteronyssinus (Der p2). Thirty mice were randomly divided into three groups, namely a negative control group (a PBS group), positive control group (an asthma group) and protein Der p2 T cell fusion epitope for specific immunotherapy (SIT) group (a Der p2 T group). The extract of house dust mites (HDM) was used to establish the asthmatic models in BALB/c mice, and the PBS group was always used with PBS buffer. Thirty minutes before spray inhalation from 25 to 27 days, the mice of the Der p2 T group were respectively injected subcutaneously with the therapeutic proteins for SIT, then the serum and bronchoalveolar lavage fluid (BALF) were collected. ELISA was used to assay the levels of IFN-γ, IL-4, and IL-13 in BALF, as well as serum levels of specific IgE and IgG2a. The lung tissue sections were stained with haematoxylin and eosin (H&E) for pathological examinations. The ELISA detection revealed that the number of eosinophil in BALF of the asthmatic mice was (5.57 ± 0.64)×105/ml, which was significantly higher than that in the PBS group [(0.50 ±0.30)×105/ml, P < 0.01], the number of eosinophil in the Der p2 T immunotherapy group decreased significantly [(3.45 ± 0.36)×105/ml, P < 0.01]. The content of IFN-γ in the PBS group, asthma group and Der p2 T group were (267.00 ± 21.98), (155.80 ± 20.53) pg/ml and (234.40 ± 24.46) pg/ml respectively. Compared with the asthma group, the mice with Der p2 T vaccine specific immune treatment produced a high level of IFN-γ (P < 0.01). The content of IL-4 in the PBS group, asthma group and Der p2 T group were (23.40 ± 5.96), (53.28 ± 8.26) pg/ml and (30.00 ± 5.50) pg/ml respectively. Compared with the asthma group, the content of IL-4 in the mice of the Der p2 T treatment group was significantly lower (P < 0.01). Compared with the asthma group [(308.10 ± 28.32) pg/ml], the content of IL-13 in BALF of the mice in the Der p2 T treatment group was significantly decreased, which was [(174.50 ± 25.99) pg/ml, P < 0.01]. The content of IL-13 in the PBS group was (95.99 ± 31.14) pg/ml. The lung tissue sections showed that the lung inflammation in the p2 T Der group was significantly less than that in the asthma group, and the inflammatory cell infiltration was significantly decreased, and airway epithelial construction remodeled. The Der p2 T cell fusion epitope, which is as vaccines for specific immunotherapy with asthma models, can alleviate effectively allergic inflammation of airway and lung in the mice, and it may be used as a candidate vaccine for asthma.

Schistosoma mansoni antigens alter activation markers and cytokine profile in lymphocytes of patients with asthma

Asthma is a chronic disease characterized by airway inflammation, obstruction and hyperresponsiveness. Severe asthma affects a small proportion of subjects but results in most of the morbidity, costs and mortality associated with the disease. Studies have suggested that Schistosoma mansoni infection reduces the severity of asthma and prevent atopy. ObjectiveWe evaluated the ability of S. mansoni antigens, Sm29 and Sm29TSP-2 to modulate lymphocyte activation status in response to the allergen of the mite Dermatophagoides pteronyssinus (Der p1) in cell cultures of individuals with asthma. MethodsThirty four patients were enrolled in this study: seventeen patients with severe asthma (SA group), seventeen patients with mild asthma (MA group) and six controls with no asthma. Peripheral blood mononuclear cells (PBMC) were obtained and stimulated with Sm29 and Sm29TSP-2 in the presence or absence of Der p1. The expression of surface markers and cytokines on lymphocytes was evaluated by flow cytometry and the levels of IL-10 in the culture supernatant were determined by ELISA. ResultsThe addition of Sm29 and Sm29TSP-2 antigens to PBMC cultures from both groups of subjects with asthma stimulated with Der p1 reduced the frequency of CD4+CD25low cells whereas and increased frequency of CD4+CD25high population was observed compared to unstimulated cultures. Moreover, cultures stimulated with Sm29TSP-2 showed a reduction in the frequency of T cells expressing CD69, IFN-γ, TNF and TGF-β in the MA group and an increase in the frequency of CD4+TSLPR+ T cells in the SA group. The addition of Sm29 to the cultures reduced the frequency of CD4+CD69+ and CD4+IL-5+ T cells in all asthmatic groups, and reduced the frequency of CD4+ T cells expressing IL-13 in the MA group. The cultures stimulated with Sm29 and Sm29TSP-2 showed an increase in the level of IL-10 in the supernatants. ConclusionThese results suggest that the addition of Sm29 and Sm29TSP-2 to the cells cultures from subjects with asthma reduced cell activation markers and altered the cytokine production pattern in a way that can potentialy control the inflammatory response associated with asthma.

The Effect of Indoor Environmental Characteristics on the Detection of House Dust Mite Der p2 and Der f2 in Asthmatics

The Effect of Indoor Environmental Characteristics on the Detection of House Dust Mite Der p2 and Der f2 in Asthmatics

Modulation of the specific immune response in Balb/c mice by intranasal application of recombinant H1D2 chimera

AbstractBACKGROUNDGroup 1 and group 2 allergens from house dust mite are the major elicitors of respiratory allergic diseases and the main candidates for immunotherapy.RESULTSThe potential therapeutic role of a chimera composed of recombinant Der p 2 (D2) linked to Influenza A virus hemagglutinin 1 (H1) for intranasal application was created, expressed and tested in a mouse model. H1D2 and D2 were produced by genetic engineering in Escherichia coli and their primary structure was confirmed by mass fingerprint. Both antigens preserved IgE reactivity in immunoblot with serum from seven house dust mite allergic persons. Balb/c mice were sensitized with D2 allergen in alum and subsequently received H1D2 or D2, intranasally. The reduced levels of serum D2 specific IgE, together with the increased serum specific IgG and IgA were detected in both groups which received H1D2 and D2 intranasally. A higher level of effector CD4+CD25+ spleen lymphocytes was found only in the group of mice which received i.n. H1D2.CONCLUSIONH1D2 chimera can have therapeutic potential in Der p 2 allergic persons as dual vaccine which, beside protective allergen specific, can provide protective antibodies against Influenza A virus hemagglutinin 1. © 2016 Society of Chemical Industry

DC-SIGN promotes allergen uptake and activation of dendritic cells in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD) is a common inflammatory skin disease, concomitant with allergic reactions to allergens. However, the exact mechanisms of allergen-induced immune responses in AD are not clear. The aim of this study is to explore the role of DC-SIGN in capturing and processing glycan-containing allergens and in the subsequent DC activation and T helper cell polarization in AD patients. MethodsDC-SIGN expression on DCs from AD patients was analysed by confocal microscopy and flow cytometry. DC-SIGN binding to common allergens was determined by ELISA. Activation of monocyte-derived dendritic cells (Mo-DCs) by allergens was analysed by evaluation of pro-inflammatory cytokines production, and their impact on T-cell responses was investigated by a DC-T cell coculture. ResultsDC-SIGN expression was higher on DCs in the lesional skin of AD patients compared with that of healthy controls and was correlated with disease severity. DC-SIGN could bind to many common allergens including house dust mite allergen (Der p2) and egg white allergen (Gal d2). Mo-DCs showed measurable expression of DC-SIGN and a concentration-dependent uptake of Der p2 and Gal d2, which was inhibited by mannan and anti-DC-SIGN Abs. Der p2 and Gal d2 induced the production of pro-inflammatory cytokines, including TNF-α and IL-6, by DCs from AD patients and facilitated Th2 and Th22 cell polarization. ConclusionsBinding of common allergens by DC-SIGN on DCs may initiate allergen sensitization of AD or provoke the relapse of AD. Regulating the allergen-DC-SIGN interaction might be a promising strategy to prevent or intervene in the progress of AD.

Electrochemical aptasensor for detecting Der p2 allergen using polycarbonate-based double-generation gold nanoparticle chip

In this study, a novel aptamer-based impedimetric biosensor for detecting the group 2 allergen of Dermatophagoides pteronyssinus (Der p2) was developed. First, an anodic aluminum oxide (AAO) membrane was prepared. A modified AAO barrier-layer surface with an array of nanohemispheres of 400nm in diameter was used as a template for the nanoelectroforming of a nickel mold. After electroforming, the AAO template was etched and a nickel nanomold with a concave nanostructure array was produced. The formed nanostructured nickel nanomold was then used in the replica molding of a nanostructured polycarbonate (PC) substrate via hot embossing. Finally, a gold thin film was sputtered onto the PC substrate to form a double-generation gold nanoparticle electrode (array of nanohemispheres with smaller nanoparticles orderly distributed on each nanohemisphere). After immobilizing specifically designed aptamers on the fabricated electrode, electrochemical impedance spectroscopy was used to determine the concentration of Der p2. The sensitivity of the proposed scheme for the detection of the dust mite antigen Der p2 was 2.088Ω/(ng/mL×cm2) with a dynamic detection range of 27.5–400ng/mL and detection limit of 16.47ng/mL.The aptamer-based impedimetric biosensor proposed in this study possesses many advantages such as high sensitivity, low cost, and high consistency over currently used sensors. The proposed sensor was found to be useful for the rapid detection of rare molecules present in an analyte.

A CCL24-dependent pathway augments eosinophilic airway inflammation in house dust mite-challenged Cd163(-/-) mice.

CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163−/− mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163−/− mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides ptyeronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163−/− mice had the same phenotype as HDM-challenged Cd163−/− mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163−/− mice, but not BMMΦs from WT mice. Lastly, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ’s from Cd163−/− mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163−/− mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.

Development of House Dust Mite Vaccine.

Mucosal vaccine based on lactic acid bacteria is an attractive strategy for prevention and treatment of allergic diseases. Here we describe the development of recombinant Lactococcus lactis expressing house dust mite (HDM) allergen as an oral vaccine. The major HDM allergen Der p2 is first codon optimized and synthesized to achieve the maximum expression level in L. lactis. After double digested by NcoI and XbaI, the derp2 fragment is ligated to the same double-digested pNZ8148 vector. The ligation is transformed to L. lactis NZ9000 and correct transformant is verified by sequencing. Western blot analysis is employed to confirm Derp2 expression in L. lactis after nisin induction.

Presence of commensal house dust mite allergen in human gastrointestinal tract: a potential contributor to intestinal barrier dysfunction

BackgroundAbnormal gut barrier function is the basis of gut inflammatory disease. It is known that house dust mite (HDM) aero-allergens induce inflammation in respiratory mucosa. We have recently reported allergen...

PLGA-Der p1 Vaccine Inhibited Tumor Growth in a Murine Model of Lung Cancer

We undertook this study to investigate the influence of PLGA-Dermatophagoides protein 1 (Der p1) vaccine on the growth of Lewis lung cancer (LLC) cells in C57/BL/6 mice. After subcutaneous transplantation of LLC, cells were injected into the axilla of C57/BL/6 mice. Five mice received intraperitoneal (i.p.) injection of PLGA-Der p1 vaccine, and another five mice received i.p. injection of PBS or PLGA as control. Body weight of C57/BL/6 mice and tumor growth were measured. Morphological change of tumor was observed under optical microscope. The expression of Ki67 and CD34 protein was detected by immunohistochemistry analysis. The concentration of IL-4 and IFN-γ in the splenocyte culture medium was detected by ELISA. The growth of transplanted tumor was inhibited markedly by PLGA-Der p1 vaccine. The protein level of Ki67 and CD34 was significantly lower in PLGA-Der p1 vaccine group than in the control group (p<0.05). The level of IFN-γ of the splenocyte culture medium was significantly higher in PLGA-Der p1 vaccine group than that in the control group (p<0.05). However, the level of IL-4 in the splenocyte culture medium was obviously lower in PLGA-Der p1 vaccine group than that in the control group (p<0.05). PLGA-Der p1 vaccine has a significant inhibitory effect on the growth of LLC cells in C57/BL/6 mice by inducing the production of a Th1 cytokine profile.

Mucosal delivery of allergen peptides expressed by Lactococcus lactis inhibit allergic responses in a BALB/c mouse model.

Allergen-specific immunotherapy (SIT) is considered to be the only curative treatment of allergy, but its safety is always affected by immunologic properties and quality of allergen. Recombinant allergen derivative could be a potential therapeutic strategy, but clinical studies showed that macromolecular derivatives could not avoid T cell-mediated side effects. In this study, five Der p2-derived peptides (DPs) containing major T cell epitopes of Der p2 were first artificially synthesized. Compared with Der p2 macromolecular derivative DM, these DPs not only fully eliminated IgE-binding capacity but also reduced T cells reactivity, suggesting these DPs could be better therapeutic molecules. For their application in vivo, Lactococcus lactis was engineered to express these DPs, and their protective effects were evaluated in BALB/c mice models. Western blot showed that all DPs could be produced in the recombinant strains. Mucosal delivery of these strains could inhibit Der p2-induced allergic responses in Der p2-sensitized mice, characterized by a reduction in specific IgE antibody and lung inflammatory responses. These protective effects were associated with an increase of specific IgG2a in serum and regulatory T cells in the mesenteric lymph nodes. On the whole, the suppressive effect induced by the DP mixture could be better than single DP, but a bit weaker than DM. These DPs could be promising candidate molecules for active vaccination and induction of tolerance, and thus promote the development of non-allergenic peptide in the treatment and prevention of allergy.

CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.

Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Concentration and determinants of molds and allergens in indoor air and house dust of French dwellings

Molds and allergens are common indoor biocontaminants. The aims of this study were to assess the concentrations of common molds in indoor air and floor dust and the concentrations of house dust mite, cat and dog allergens in mattress dust in French dwellings, and to assess predictors of these concentrations. A sample of 150 houses in Brittany (western France) was investigated. Airborne Cladosporium and Penicillium were detected in more than 90% of the dwellings, Aspergillus in 46% and Alternaria in only 6% of the housings. Regarding floor dust samples, Cladosporium and Penicillium were detected in 92 and 80% of the housings respectively, Aspergillus in 49% and Alternaria in 14%. House dust mite allergens Der p1 and Der f1 were detected in 90% and 77% of the mattress dust samples respectively and Can f1 and Fel d1 in 37% and 89% of the homes. Airborne and dustborne mold concentrations, although not statistically correlated (except for Aspergillus) shared most of their predictors. Multivariate linear models for mold levels, explaining up to 62% of the variability, showed an influence of the season, of the age of the dwelling, of aeration habits, presence of pets, smoking, signals of dampness, temperature and relative humidity. Allergens in the dust of the mattress were strongly related to the presence of pets and cleaning practices of bedsheets, these factors accounting for 60% of the variability. This study highlights ubiquitous contamination by molds and underlines complex interaction between outdoor and indoor sources and factors.

Different anti-apoptotic effects of house dust mite allergen on eosinophil apoptosis between atopic and non-atopic asthmatic subjects

House dust mite is a major allergen in allergic diseases such as asthma. In this study, we investigated the effects of house dust mite on constitutive eosinophil apoptosis in normal and asthmatic subjects. We classified asthmatic subjects into those with atopic and non-atopic asthma depending on the presence of DP-specific IgE or/and DF-specific IgE in serum. Both blood and bronchoalveolar lavage fluid (BALF) eosinophils in atopic asthma were elevated when compared with normal and non-atopic eosinophils. Dermatophagoides pteronissinus extract (DP) inhibited constitutive eosinophil apoptosis of atopic asthmatic subjects, but not that of normal and non-atopic subjects. DF had no effect on eosinophil apoptosis in normal and asthmatic subjects. The anti-apoptotic effects of DP were not altered by E64, a cysteine protease inhibitor, or aprotinin, a serine protease inhibitor, and Der p1 and Der p 2 had no effect on eosinphil apoptosis of normal and asthmatic patients, including atopic and not-atopic patients. Anti-apoptotic signaling mediated by DP in atopic asthma was associated with the TLR4/PI3K/Akt/ERK/NF-κB pathway. Activation of procaspase 3 and procaspase 9 was delayed by DP stimulation. Our results indicate that DP induces eosinophilic inflammation by inhibiting eosinophil apoptosis, and that the inhibitory effect is associated with exposure of asthma subjects to DP. A better understanding of the difference between atopic and non-atopic asthma will help elucidate the pathogenesis and develop methods for treatment of asthma.

An Exploratory Pilot Study of Genetic Marker for IgE-Mediated Allergic Diseases with Expressions of FcεR1α and Cε.

The high affinity immunoglobulin E (IgE) receptor-FcεR1 is mainly expressed on the surface of effector cells. Cross-linking of IgE Abs bound to FcεR1 by multi-valent antigens can induce the activation of these cells and the secretion of inflammatory mediators. Since FcεR1 plays a central role in the induction and maintenance of allergic responses, this study aimed to investigate the association of FcεR1 with the allergic phenotype of Cε expression and cytokine and histamine release from peripheral leukocytes. Peripheral leukocytes from 67 allergic and 50 non-allergic subjects were used for genotyping analysis. Peripheral mononuclear cells (PBMCs) were used for Cε expression and ELISpot analysis, while polymorphonuclear cells (PMNs) were used for histamine release. The association between genotype polymorphism of the FcεR1α promoter region (rs2427827 and rs2251746) and allergic features of Cε expression and histamine were analyzed, and their effects on leukocytes function were compared with wild type. The genotype polymorphisms of FcεR1α promoter region with CT and TT in rs2427827 and TC in rs2251746 were significantly higher in allergic patients than in non-allergic controls. Patients with single nucleotide polymorphism (SNP) of FcεR1α promoter region had high levels of total IgE, mite-specific Der p 2 (Group 2 allergen of Dermatophagoides pteronyssinus)-specific IgE and IgE secretion B cells. The mRNA expression of FcεR1α was significantly increased after Der p2 stimulation in PBMCs with SNPs of the FcεR1α promoter region. Despite the increased Cε mRNA expression in PBMCs and histamine release from PMNs and the up-regulated mRNA expression of interleukin (IL)-6 and IL-8 secretions after Der p2 stimulation, there was no statistically significant difference between SNPs of the FcεR1α promoter region and the wild type. SNPs of FcεR1α promoter region were associated with IgE expression, IgE producing B cells, and increased Der p2-induced FcεR1α mRNA expression. These SNPs may be used as a disease marker for IgE-mediated allergic inflammation caused by Dermatophagoides pteronyssinus.

Early-life house dust mite allergens, childhood mite sensitization, and respiratory outcomes.

Exposure to indoor allergens during early life may play a role in the development of the immune system and inception of asthma. To describe the house dust mite (HDM) allergen concentrations in bedroom dust during early life and to evaluate its associations with HDM sensitization, wheezing, and asthma, from birth to school age, in 5 geographically spread European birth cohorts. We included 4334 children from INMA-Menorca (Spain), BAMSE (Sweden), LISAplus and MAS (Germany), and PIAMA-NHS (the Netherlands). Dust samples were collected from bedrooms during early life and analyzed for Dermatophagoides pteronyssinus (Der p1) and Dermatophagoides farinae (Der f1). HDM concentrations were divided into four categories. Sensitization was determined by specific IgE. Wheezing and asthma information up to 8/10 years was collected through questionnaires. We performed mixed-effects logistic regression models and expressed associations as odds ratios with 95% confidence intervals. House dust mite concentrations varied across cohorts. Mean allergen concentrations were highest in INMA-Menorca (geometric mean (GM) Der p1 = 3.3 μg/g) and LISAplus (GM Der f1 = 2.1 μg/g) and lowest in BAMSE (GM Der p1 = 0.1 μg/g, Der f1 = 0.3 μg/g). Moderate and high HDM concentrations were significantly (P-values < 0.05) associated with 50-90% higher prevalence of HDM sensitization. No significant associations were observed with respiratory outcomes. Our study based on geographically spread regions, a large sample size, and a wide range of allergen concentration shows that HDM allergen concentrations vary across regions and that exposure during early life plays a role in the development of allergic sensitization but not in the development of respiratory outcomes.

Anaphylaxis caused by flaxseed

Methods and results Prick-prick carried out with flaxseed showed a strong positivity. Skin prick test to inhaled allergens were positive to mites, dog and cat danders, plain tree and grasses. Skin prick test to food allergens were positive to almond, hazelnut, peanut, chestnut, walnut, mustard, lentils, soybeans, rice, oat, corn and lupine. Total IgE was: 291 UI/ml. Specific IgE (CAP, ThermoFisher Scientific) to flaxseed was 2.68 kU peach 7.65 kU/L, peanut 2.01 kU / L , lettuce 0.92 kU / L , lupin 3.76 kU / L. Corn IgE 2.71 kU / L , Pru p3 7.62 kU / L , Cor a8 0.68 kU / L. ISAC, ImmunoCAP was positive to Fel d1, Phl p1, Der f1, Der f2, Der p1, Der p2, Pla a3, Ara h9, Cor a8, Jug r3, Pru p3 and Art v3. ISAC performed after inhibit patients’s serum (ISAC inhibition) with a complete flaxseed extract (f233:Linum usitatissium from ImmunoCAP, ThermoFisher Scientific) did not reduced the positivity to non-specific lipid transfer proteins (nsLTP). ISAC inhibition with a complete mite extract (d1: D. pteronyssinus from ImmunoCAP, ThermoFisher Scientific) reduces exclusively the signal of mites (Der f1, Der f2, Der p1, Der p2) in >83%.An immunoblot performed with flaxseed extracts (soluble and liposoluble), showed the presence of an IgE-binding band of about 18 kDa in the liposoluble fraction and a 20 kDa band in the soluble one.

Suppression of dust mite allergy by mucosal delivery of a hypoallergenic derivative in a mouse model.

Allergic asthma caused by house dust mite (HDM) is becoming a public health problem. Specific immunotherapy is considered to be the only curative treatment, but it is always associated with IgE-mediated side effects in the therapy process. A few studies showed that the disruption of allergen IgE epitopes could reduce IgE reactivity and thus reduce allergenic activity. In this study, a hypoallergenic derivative of the major HDM allergen Der p2 was constructed by genetic engineering. This derivative was confirmed to have a considerably reduced IgE reactivity compared with Der p2. For its application in vivo, recombinant Lactococcus lactis (LL-DM) was engineered to deliver the Der p2 derivative to the intestinal mucosal surface. Oral administration of LL-DM significantly alleviated Der p2-induced airway inflammation, as shown by reduced inflammatory infiltration and a reduction in Th2 cytokines in bronchoalveolar lavage. This protective effect was associated with an up-regulation of specific IgG2a and a decrease in IL-4 level in the spleen which may affect specific IgE response. Moreover, the levels of regulatory T cells in the mesenteric lymph nodes and spleen were markedly increased in mice fed with LL-DM, suggesting that LL-DM can inhibit allergic responses via the induction of regulatory T cell. Our results indicate that the Der p2 derivative is a promising therapeutic molecule for specific immunotherapy and recombinant lactic acid bacteria could be developed as a promising treatment or prevention strategy for allergic diseases.

Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling.

PurposeHouse-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation.MethodsInternalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA).ResultsInternalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion.ConclusionsHuman airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.