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House dust mite (HDM) is a globally ubiquitous domestic cause of allergic diseases. There is a pressing demand to discover efficient, harmless, and eco-friendly natural extracts to inhibit HDM allergens that are more likely to trigger allergies and challenging to be prevented entirely. This study, therefore, is aimed at assessing the inhibition of the allergenicity of major HDM allergen Der f 2 by todomatsu oil extracted from residues of Abies Sachalinensis . The inhibition was investigated experimentally (using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) and in silico using molecular docking. The results showed that todomatsu oil inhibits the allergenicity of Der f 2 by reducing its amount instead of the IgG binding capacity of a single protein. Moreover, the compounds in todomatsu oil bind to Der f 2 via alkyl hydrophobic interactions. Notably, most compounds interact with the hydrophobic amino acids of Der f 2, and seven substances interact with CYS27. Contrarily, the principal compounds fail to attach to the amino acids forming the IgG epitope in Der f 2. Interestingly, chemical components with the lowest relative percentages in todomatsu oil show high-affinity values on Der f 2, especially β-maaliene (-8.0 kcal/mol). In conclusion, todomatsu oil has been proven in vitro as a potential effective public health strategy to inhibit the allergenicity of Der f 2.

Nineteen U.S. allergen extracts were standardized by the U.S. Food and Drug Administration (FDA) between 1987 and 1998, including of two house-dust mites, short ragweed, cat hair and cat pelt, seven temperate and one southern grass, and six Hymenoptera venom preparations. Relevant literature was reviewed. For each allergen, a "representative" extract was established; the potency of each representative extract was determined by measurement of the total protein content (Hymenoptera venom), radial diffusion measurement of the dominant allergen (short ragweed and cat), or, if there was no dominant allergen, then by quantitative skin testing by using the ID 50 EAL (intradermal dilution for 50 mm sum of erythema determines the bioequivalent allergy units) method. In vitro tests were developed to allow the manufacturer to demonstrate that each lot of its extract was statistically identical, within defined limits, to the FDA reference extract. These tests included radial immunodiffusion, competitive enzyme-linked immunosorbent assay, and isoelectric focusing. The standardized extracts offer the advantage of consistent potency from lot to lot for each manufacturer and also from manufacturer to manufacturer, and assure the presence of recognized significant allergens within the extract. Therefore, standardized extracts offer improved safety and efficacy over their nonstandardized predecessors.

Understanding the functional potential of the gut microbiome is of primary importance for the design of innovative strategies for allergy treatment and prevention. Here we report the gut microbiome features of 90 children affected by food (FA) or respiratory (RA) allergies and 30 age-matched, healthy controls (CT). We identify specific microbial signatures in the gut microbiome of allergic children, such as higher abundance of Ruminococcus gnavus and Faecalibacterium prausnitzii, and a depletion of Bifidobacterium longum, Bacteroides dorei, B. vulgatus and fiber-degrading taxa. The metagenome of allergic children shows a pro-inflammatory potential, with an enrichment of genes involved in the production of bacterial lipo-polysaccharides and urease. We demonstrate that specific gut microbiome signatures at baseline can be predictable of immune tolerance acquisition. Finally, a strain-level selection occurring in the gut microbiome of allergic subjects is identified. R. gnavus strains enriched in FA and RA showed lower ability to degrade fiber, and genes involved in the production of a pro-inflammatory polysaccharide. We demonstrate that a gut microbiome dysbiosis occurs in allergic children, with R. gnavus emerging as a main player in pediatric allergy. These findings may open new strategies in the development of innovative preventive and therapeutic approaches. Trial: NCT04750980., (© 2021. The Author(s).)

The involvement of autophagy and its dysfunction in asthma is still poorly documented. By using a murine model of chronic house dust mite (HDM)-induced airway inflammation, we tested the expression of several autophagy markers in the lung and spleen of asthma-like animals. Compared to control mice, in HDM-sensitized and challenged mice, the expression of sequestosome-1/p62, a multifunctional adaptor protein that plays an important role in the autophagy machinery, was raised in the splenocytes. In contrast, its expression was decreased in the neutrophils recovered from the bronchoalveolar fluid, indicating that autophagy was independently regulated in these two compartments. In a strategy of drug repositioning, we treated allergen-sensitized mice with the therapeutic peptide P140 known to target chaperone-mediated autophagy. A single intravenous administration of P140 in these mice resulted in a significant reduction in airway resistance and elastance, and a reduction in the number of neutrophils and eosinophils present in the bronchoalveolar fluid. It corrected the autophagic alteration without showing any suppressive effect in the production of IgG1 and IgE. Collectively, these findings show that autophagy processes are altered in allergic airway inflammation. This cellular pathway may represent a potential therapeutic target for treating selected patients with asthma.

Objective: To present an update of birth cohort study designs and their contributions to allergic risk., Data Sources: The PubMed database was used to search for relevant articles., Study Selections: Peer-reviewed prospective and retrospective studies involving the assessment of allergy using human birth cohorts between 2014 and 2021 were evaluated., Results: Parental history of allergic diseases, especially in cases involving both parents, is associated with increased risk of allergy. Exposure to prenatal and postnatal smoking and limited diet diversity were associated with increased allergic burden. The impact of early-life infections and antibiotics on disease development may be associated with the onset of asthma, though this remains debated. Cohort studies also revealed that the mode of delivery and breastfeeding duration affect the odds ratio of asthma and eczema development. Household exposures, including pets, house dust mites, and scented aeroallergens may confer protective effects, whereas high air pollution exposure and low socioeconomic status may be risk enhancing. Exposure to antibiotics during early life may be associated with increased asthma risk, whereas viral infections may lead to disease protection, though the impact of the coronavirus disease 2019 pandemic on allergic risk is yet to be understood., Conclusion: Although evaluating the risk of allergic disease development is complex, clinicians can apply these insights on the multifactorial nature of atopy to better understand and potentially mitigate disease development., (Copyright © 2021. Published by Elsevier Inc.)

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [ Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b + platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b + platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Mouse models of allergic asthma have been utilized to establish the role of T helper type 2 (Th2) cells in driving lung inflammation, airway hyperresponsiveness, and obstruction. Here, we present the allergic asthma models, in which mice are hypersensitized to ovalbumin (OVA) and house dust mite (HDM). These models mimic the major characteristics of human asthma including the eosinophilic inflammation and hyperactivity of the airway, overproduction of Th2 cytokines in the lung, and elevated total and allergen-specific immunoglobulin E (IgE) in serum.

Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. As a consequence of the IgE production, subsequent allergen reexposure results in a classic allergic response with distinct early and late phases, both resulting in bronchoconstriction and shortness of breath. Allergen-specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma. Both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have shown clinical efficacy in long-term suppression of the allergic response. Although AIT treatments are very successful for rhinitis, application in asthma is hampered by variable efficacy, long duration of treatment, and risk of severe side effects. A more profound understanding of the mechanisms by which AIT induces tolerance to allergens in sensitized individuals is needed to be able to improve its efficacy. Mouse models have been very valuable in preclinical research for characterizing the mechanisms of desensitization in AIT and evaluating novel approaches to improve its efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen adsorbed to aluminum hydroxide, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of allergen extracts as an immunotherapy treatment. Finally, mice are challenged by intranasal allergen administrations. We will also describe the protocols as well as the most important readout parameters for the measurements of invasive lung function, serum immunoglobulin levels, isolation of bronchoalveolar lavage fluid (BALF), and preparation of cytospin slides. Moreover, we describe how to perform ex vivo restimulation of lung single-cell suspensions with allergens, flow cytometry for identification of relevant immune cell populations, and ELISAs and Luminex assays for assessment of the cytokine concentrations in BALF and lung tissue.

House dust mite (HDM) is an environmental allergen ubiquitously present indoors, causing allergic inflammation in dogs. However, it is unclear whether HDM allergens can be detected in the gastrointestinal (GI) tract of dogs. In addition, although expression of interleukin (IL)-1β is increased in the intestinal mucosa of dogs with chronic enteropathy (CE), the role of HDM allergens in the production of IL-1β has not been evaluated. The objectives of this study were to determine the presence of HDM allergens in the GI tract of dogs and to elucidate the effect of HDM on IL-1β expression in canine macrophages. HDM allergen, Dermatophagoides pteronyssinus (Der p) 1, was quantified in the gastric and duodenal fluids and the duodenal and colonic mucosae of dogs with CE and healthy laboratory dogs, and faeces of dogs with CE, healthy laboratory dogs and healthy client-owned dogs. Gene expression and protein levels of IL-1β were measured in HDM-stimulated canine peripheral macrophages from healthy laboratory dogs. Der p 1 was detected in the gastric and duodenal fluids of dogs with CE and healthy laboratory dogs, and faeces of all dogs examined. Der p 1 levels in the duodenal and colonic mucosae were significantly higher in dogs with CE than in healthy laboratory dogs. HDM increased both gene expression and protein levels of IL-1β in canine macrophages. These findings demonstrate the presence of HDM allergens in the GI tract of dogs and the possible involvement of HDM allergens in the pathogenesis of CE by promoting IL-1β expression in macrophages., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Characteristic of allergic asthma, CD4+Th2 lymphocytes secrete Th2 cytokines, interleukin (IL)-4, IL-13, and IL-5 that mediate the inflammatory immune response. Surface expression of CD2 and its ligand, CD58, is increased on the monocytes and eosinophils of asthma patients, which correlate with elevated serum IgE levels, suggesting that CD2 may contribute to allergic airway inflammation. Using a murine model of asthma, we observed that house dust mice extract (HDME)-exposed Balb/c mice have increased airway hyperresponsiveness (AHR), lung inflammation, goblet cell hyperplasia, and elevated levels of Th2 cytokines in the lungs, as well as increased serum IgE levels as compared to the control mice. In contrast, with the exception of serum IgE levels, all the other parameters were significantly reduced in HDME-treated Cd2 -/- mice. Interestingly, Il13 but not Il4 or Il5 gene expression in the lungs was dramatically decreased in HDME-exposed Cd2 -/- mice. Of note, the gene expression of IL-13 downstream targets (Muc5b and Muc5ac) and high affinity IL-13Rα2 were upregulated in the lungs of HDME-exposed Balb/c mice but were significantly reduced in HDME-exposed Cd2 -/- mice. Consistently, gene expression of microRNAs regulating mucin production, inflammation, airway smooth muscle cell proliferation and IL-13 transcripts were increased in the lungs of HDME-exposed Cd2 -/- mice. Given the established role of IL-13 in promoting goblet cell hyperplasia, lung inflammation and AHR in allergic asthma, our studies reveal a unique role for CD2 in the regulation of Th2-associated allergic asthma., (Copyright © 2020 Hashem, Kammala, Thaxton, Griffin, Mullany, Panettieri, Subramanian and Das.)

We have previously demonstrated that upregulation of Sonic hedgehog (SHH) expression in allergic airway epithelia essentially contributes to the goblet cell metaplasia and mucous hypersecretion. However, the mechanism underlying the upregulation of SHH expression remains completely unknown. In cultured human airway epithelial cells, IL-4/IL-13 but not IL-5 robustly induces the mRNA and protein expression of SHH and in turn activates SHH signaling by promoting the JAK/STAT6-controlling transcription of SHH gene. Moreover, intratracheal instillation of IL-4 and/or IL-13 robustly activates STAT6 and concomitantly upregulates SHH expression in mouse airway epithelia, whereas, in Club cell 10-kDa protein (CC10)-positive airway epithelial cells of children with asthma, activated STAT6 closely correlates with the increased expression of SHH and high activity of SHH signaling. Finally, intratracheal inhibition of STAT6 by AS-1517499 significantly diminished the allergen-induced upregulation of SHH expression, goblet cell phenotypes, and airway hyperresponsiveness, in an ovalbumin- or house dust mite-induced mouse model with allergic airway inflammation,. Together, upregulation of SHH expression by IL-4/IL-13-induced JAK/STAT6 signaling contributes to allergic airway epithelial remodeling, and this study thus provides insight into how morphogen signaling is coordinated with Th2 cytokine pathways to regulate tissue remodeling in chronic airway diseases.

The hyperconstriction of airway smooth muscle (ASM) is the main driving mechanism during an asthmatic attack. The airway lumen is reduced, resistance to airflow increases, and normal breathing becomes more difficult. The tissue contraction can be temporarily relieved by using bronchodilator drugs, which induce relaxation of the constricted airways. In vitro studies indicate that relaxation of isolated, precontracted ASM is induced by mechanical oscillations in healthy subjects but not in asthmatic subjects. Further, short-term acute asthmatic subjects respond to superimposed pressure oscillations (SIPO) generated in the range of 5-15 Hz with ~50% relaxation of preconstricted sensitized airways. Mechanical oscillations, and specifically SIPO, are not widely characterized in asthmatic models. The objective of this in vivo study is to determine the effects of a range of oscillation patterns similar to our previous acute study differing from normal breathing. Both healthy and sensitized mice were observed, with their responses to SIPO treatments measured during induced bronchoconstriction resulting from acetylcholine (Ach) challenge. SIPO-generated results were compared with data from treatments using the bronchorelaxant isoproterenol (ISO). The study shows that SIPO in the range of 5-20 Hz induces relaxation in chronic sensitized airways, with significant improvements in respiratory parameters at SIPO values near 1.7 cmH 2 O irrespective of the frequency of generation.

The incidence of asthma has increased from 5.5% to near 8% of the population, which is a major health concern. The hallmarks of asthma include eosinophilic airway inflammation that is associated with chronic airway remodeling. Allergic airway inflammation is characterized by a complex interplay of resident and inflammatory cells. MicroRNAs (miRNAs) are small noncoding RNAs that function as posttranscriptional modulators of gene expression. However, the role of miRNAs, specifically miR-451, in the regulation of allergic airway inflammation is unexplored. Our previous findings showed that oxidant stress regulates miR-451 gene expression in macrophages during an inflammatory process. In this paper, we examined the role of miR-451 in regulating macrophage phenotype using an experimental poly-allergenic murine model of allergic airway inflammation. We found that miR-451 contributes to the allergic induction of CCL17 in the lung and plays a key role in proasthmatic macrophage activation. Remarkably, administration of a Sirtuin 2 (Sirt2) inhibitor diminished alternate macrophage activation and markedly abrogated triple-allergen [dust mite, ragweed, Aspergillus fumigatus (DRA)]-induced lung inflammation. These data demonstrate a role for miR-451 in modulating allergic inflammation by influencing allergen-mediated macrophages phenotype.

Background: Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated., Objectives: We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma., Methods: After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM + HDM or vehicle and analysed., Results: In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM + HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2., Conclusions & Clinical Relevance: TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident in the in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma., (© 2019 John Wiley & Sons Ltd.)

Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions. Here we report the immuno-characterization of its group 2 allergen, Blo t 2. Allergen Blo t 2 was amplified from the cDNA of B. tropicalis using degenerate primers, expressed in Escherichia coli as a recombinant protein and purified to homogeneity. The mature protein of Blo t 2 was 126 amino acids long with 52% sequence identity to Der p 2 and apparent molecular mass of 15 kDa. Circular dichroism spectroscopy showed that Blo t 2 is mainly a beta-sheeted protein. We confirmed the presence of three disulfide bonds in recombinant (r) Blo t 2 protein using electrospray mass spectrometry. Thirty-four percent of dust-mite allergic individuals from the Singapore showed specific IgE binding to rBlo t 2 as tested using immuno dot-blots. IgE-cross reactivity assays showed that Blo t 2 had between 20-50% of unique IgE-epitopes compared to Der p 2. IgE binding of native and recombinant forms of Blo t 2 were highly concordant (r 2  = 0.77, p < 0.0001) to rBlo t 2. Dose-dependent in vitro histamine was observed when rBlo t 2 was incubated with whole blood of Blo t 2 sensitized individuals, demonstrating that it is a functional allergen. Nine naturally occurring isoforms of Blo t 2 were identified in this study, each having between 1-3 amino acid variations compared to the reference clone. Blo t 2 is a clinically relevant allergen of B. tropicalis as it has unique IgE epitopes compared to major group 2 allergens from Dermatophagoides spp.

Background: House dust mites are important allergen sources and some of these allergenic proteins may contain carbohydrate moieties, which are able to be isolated using lectins, as Concanavalin A (ConA). This study aimed to investigate allergenicity (IgE) and antigenicity (IgG1 and IgG4) of ConA-unbound and ConA-bound Dermatophagoides pteronyssinus (Dpt) crude extracts using sera of mite-allergic patients as well as inhibition capacity of antibody binding., Material and Methods: We obtained mannose-enriched and mannose-depleted fractions from Dpt by ConA affinity chromatography. Both ConA-bound and ConA-unbound fractions were evaluated by ELISA and Western Blotting for specific IgE, IgG1, and IgG4 reactivity with sera obtained from 95 mite-allergic patients (DP+) and 92 nonallergic (NA) subjects. Inhibition ELISA was used to assess cross-reactivity between Dpt extract and its fractions., Results: Among the DP+ patients, no difference was found between ConA-unbound and ConA-bound fractions regarding the levels of specific IgE, IgG1, and IgG4. Nonallergic subjects had the same levels of specific IgG1 to both ConA-unbound and ConA-bound fractions, although for specific IgG4, values were higher for ConA-bound. A positive correlation was found among specific IgE, IgG1, and IgG4 levels when Dpt was compared to ConA-unbound and ConA-bound fractions. Recognition of crude Dpt by IgE, IgG1, and IgG4 was highly inhibited by ConA-unbound and ConA-bound fractions. Western Blotting revealed a broad spectrum of bands ranging from 14 to 116 kDa recognized by specific IgE and IgG4. However, IgG1 reached higher frequency values on high molecular weight polypeptides., Conclusion: ConA-unbound and ConA-bound fractions derived from D. pteronyssinus crude extract revealed important components involved in the IgE recognition in allergic patients as well as IgG1 and/or IgG4 in allergic and healthy subjects., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper.

This study was conducted to determine whether exposure to particulate matter 2.5 (PM 2.5 ) affects the immune tolerance of neonatal mice via the regulation of PD-L1 expression. One-week-old BALB/c mice were exposed to PM 2.5 for 8 days. From day 8 to day 18, the mice were treated with 5 μg house dust mite (HDM) (i. n.) every two days. Adenovirus-carried PD-L1 overexpression vectors were infected into mice via nasal inhalation 6 days after exposure to PM 2.5 . Airway hyperresponsiveness (AHR) was examined in mice 19 days after exposure to PM 2.5 , and the related parameters of airway inflammation were studied on day 22. Co-exposure to PM 2.5 and HDM reduced PD-L1 expression but greatly increased infiltration of inflammatory cells, which was reversed by PD-L1 overexpression. Co-exposure to PM 2.5 and HDM also elevated serum IL-4, IL-5 and IL-13 levels and reduced TGF-β level. Exposure to PM 2.5 alone slightly increased the numbers of dendritic cells (DCs) but reduced the numbers of antigen-presenting cells expressing PD-L1 and Treg cells. Therefore, early exposure to PM 2.5 reduced PD-L1 expression in the lungs of neonatal mice, which interfered with immune tolerance establishment and subsequently resulted in allergic airway inflammation.

Background: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response., Objective: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells., Methods: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro., Results: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway., Conclusions and Clinical Relevance: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state., (© 2018 John Wiley & Sons Ltd.)

Early life exposure to fine particulate matter (PM) in air is associated with infant respiratory disease and childhood asthma, but limited epidemiological data exist concerning the impacts of ultrafine particles (UFPs) on the etiology of childhood respiratory disease. Specifically, the role of UFPs in amplifying Th2- and/or Th17-driven inflammation (asthma promotion) or suppressing effector T cells (increased susceptibility to respiratory infection) remains unclear. Using a mouse model of in utero UFP exposure, we determined early immunological responses to house dust mite (HDM) allergen in offspring challenged from 0 to 4 wk of age. Two mice strains were exposed throughout gestation: C57BL/6 (sensitive to oxidative stress) and BALB/C (sensitive to allergen exposure). Offspring exposed to UFPs in utero exhibited reduced inflammatory response to HDM. Compared with filtered air (FA)-exposed/HDM-challenged mice, UFP-exposed offspring had lower white blood cell counts in bronchoalveolar lavage fluid and less pronounced peribronchiolar inflammation in both strains, albeit more apparent in C57BL/6 mice. In the C57BL/6 strain, offspring exposed in utero to FA and challenged with HDM exhibited a robust response in inflammatory cytokines IL-13 and Il-17. In contrast, this response was lost in offspring exposed in utero to UFPs. Circulating IL-10 was significantly up-regulated in C57BL/6 offspring exposed to UFPs, suggesting increased regulatory T cell expression and suppressed Th2/Th17 response. Our results reveal that in utero UFP exposure at a level close to the WHO recommended PM guideline suppresses an early immune response to HDM allergen, likely predisposing neonates to respiratory infection and altering long-term pulmonary health., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: House dust mite (HDM) is the major source of indoor allergens that cause airway disease. Recent evidence suggests that Gram-negative/positive bacteria produce nano-sized extracellular vesicles (EVs) containing diverse components, including various immunostimulatory molecules. However, the association between bacteria-derived EVs and development of airway disease is unclear., Objective: To identify and isolate HDM-derived EVs and to evaluate their effect on the development of airway inflammation., Methods: Extracellular vesicles were isolated from crude HDM extracts by ultra-centrifugation, and their physical and immunological characteristics and roles in airway inflammation were tested in vitro and in murine models of airway inflammation. In addition, 16s metagenome analysis of nucleic acid from EVs was performed to identify their origin., Results: Round, bilayered vesicles measuring 80-100 nanometres and containing abundant amounts of LPS were isolated. These vesicles induced innate immune responses both in vitro and in vivo. Intranasal exposure of naïve mice to HDM EVs induced production of cytokines associated with development of Th2-mediated and mixed (Th1-/Th2-/Th17-mediated) airway inflammation to allergen. Metagenome analysis identified Bacteroidetes and Proteobacteria as the probable sources of HDM EVs., Conclusion: House dust mite EVs originating from Gram-negative bacteria may play an important role on the development of airway inflammation., (© 2018 John Wiley & Sons Ltd.)

Aims: Atopic dermatitis (AD) is a chronic inflammatory skin disease, with a steadily increasing prevalence. Lactic acid bacteria (LAB) have been widely used in the food industry and are an attractive option for preventing and treating allergic skin diseases. We previously isolated new LABs including Lactococcus lactis KR-050L from Gajuknamu kimchi, and showed the anti-inflammatory effects of extract of L. lactis KR-050L culture broth (LLK). In this study, we investigated the effects of LLK on AD., Methods and Results: For the in vitro study, we used human keratinocytes (HaCaT) and mast cells (RBL-2H3). In vivo study, we investigated the effects of LLK on Dermatophagoides farinae extract (DFE) and 2,4-dinitrochlorobenzene (DNCB)-induced atopic skin inflammation in mice. LLK suppressed expression of pro-inflammatory cytokines and chemokines by down-regulation of p38 MAPK, STAT1 and nuclear translocation of NF-κB in keratinocytes. Topical application of LLK suppressed AD symptoms based on reduction in ear thickness, serum IgE levels and immune cell infiltration. Furthermore, LLK inhibited serum histamine levels and mast cells infiltration in vivo, and reduced mast cells activation in vitro., Conclusions: These results suggest that LLK inhibits AD symptoms through inhibition of keratinocytes and mast cells activation., Significance and Impact of the Study: LLK is a potential therapeutic candidate for AD treatment., (© 2018 The Society for Applied Microbiology.)

Background: The dust mite Dermatophagoides farinae is a common worldwide cause of indoor allergies induced by its proteins, including the mid-tier allergen Der f 7., Objective: To identify conformational epitopes in Der f 7 using mimotope mapping and computational modelling., Methods: Here, we used standard hybridoma technology to generate 3 new monoclonal antibodies against Der f 7 and performed mimotope mapping by probing a random peptide phage display library. Computational tools, including Minox and the DiscoTope-2.0 Server were used to assess the structure and potential position of antigenic residues within Der f 7., Results: Thirteen mimotopes sharing the common sequence --XX[LST]P[-E][LI]MLPLR[-S]- were identified. Further, computationally-predicted conformational epitopes were found at residues 1-7, 10, 27, 76-81, 92, and 130-133 of Der f 7, and the key amino acids for these epitopes were deduced to be 2P, 3I, 10E, 27E, 78E, 79E, 81I, 130S, and 132E based on the common mimotope sequence., Conclusion: We identified Der f 7 peptide mimotopes that may model binding sites for blocking antibodies. These may guide the development of immunotherapy for individuals with hypersensitivity to Der f 7., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

T helper 17 (Th17) cells has proven to be crucial in the pathogenesis of severe asthma. Although it is known that Suppressor of cytokine signaling 3 (SOCS3) is involved in differentiation of Th17 cells but, how it affects severe asthma is uncertain. Since previous studies indicated that Methtyl-CpG binding domain protein 2 (MBD2) null mice was deficient in Th17 cell differentiation, the aim of the present study was to understand how MBD2 interacts with SOCS3 to regulate Th17 cell differentiation in severe asthma. Here, we show that SOCS3 expression was significantly decreased in Th17-mediated severe asthmatic mice, accompanied by elevated STAT3 phosphorylation and RORγt expression. Knock-down of SOCS3 promoted the differentiation of naïve T cells into Th17 cells through STAT3/RORγt pathway. Meanwhile, MBD2 was overexpressed in Th17-mediated severe asthmatic mice. Intervention of MBD2 expression lead to a negative change of SOCS3 expression, whereas the differentiation of Th17 cells showed positive change. In addition, MBD2 knockout (MBD2-KO) mice displayed increased SOCS3 expression and decreased Th17 differentiation after severe asthma modeling. Taken together, our results suggest that MBD2 might facilitate Th17 cell differentiation via down-regulating SOCS3 expression in severe asthma. These findings uncover new roles for SOCS3 and MBD2, and provide a potential target for treatment of severe asthma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Allergic rhinitis with or without conjunctivitis (AR/C) is common, necessitating evaluation of SQ house dust mite (HDM) sublingual immunotherapy (SLIT)-tablet efficacy in various subgroups., Objective: To evaluate 12 SQ-HDM efficacy and safety across subgroups, and the onset, duration, and recurrence of local application site reactions., Methods: Subgroup (age, sex, race, asthma status, and allergen sensitization) efficacy was assessed using pooled data from 2 previously described trials of daily 12 SQ-HDM vs placebo for AR/C (n = 2,138). Efficacy was measured by average total combined rhinitis score (TCRS; rhinitis daily symptom plus medication score) during the last 8 weeks of treatment. Safety in subgroups and local application site reaction onset, duration, and recurrence were evaluated using pooled data from 5 previously described trials of SQ HDM SLIT-tablet (n = 2,923)., Results: Significant (based on 95% confidence intervals [CIs]) reduction in TCRS was seen with 12 SQ-HDM relative to placebo across all subgroups, with TCRS improvements ranging from 15% to 25%. The AE profile was generally similar within subgroups. Approximately 95% of local application site reactions were mild to moderate in severity. Median duration on day 1 of treatment for the most common local application site reactions (throat irritation, oral pruritus, ear pruritus, and lip swelling) ranged from 30 to 60 minutes; median first day of onset ranged from days 1 to 4 of treatment; median days that reactions recurred ranged from 3 to 12 days., Conclusion: Treatment with 12 SQ-HDM consistently improved symptoms and was well tolerated in relevant subgroups of subjects with HDM AR/C. Local application site reactions to 12 SQ-HDM were typically mild to moderate and transient., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To provide a comprehensive overview of common school exposures and the association between school exposures and pediatric asthma morbidity., Data Sources: A comprehensive literature review was performed using PubMed., Study Selections: Full-length, peer-reviewed studies published in English were considered for review. In vivo, in vitro, and animal studies were excluded. Studies of school exposure to cockroach, mouse, dust mite, dog, cat, molds, pollution, and endotoxin associated with asthma and asthma morbidity were considered., Results: The current literature establishes an association between school exposure and pediatric asthma morbidity. There is a need for ongoing research to evaluate the effects of school-based environmental interventions on asthma morbidity., Conclusion: It is evident that the indoor school environment is a significant reservoir of allergens, molds, pollutants, and endotoxin and that there is an association between school exposure and pediatric asthma morbidity. School-based interventions have the potential for substantial individual, community, and public health benefit. It is important that researchers continue to study the health effects associated with school exposures and assess cost-effectiveness of multifaceted school-based interventions., (Copyright © 2018 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

House dust mites are typically absent in homes in arid and semiarid climates due to low humidity. Evaporative "swamp" cooling significantly increases indoor humidity in dry climates and is suspected of promoting dust mite survival in these regions. We investigated the prevalence and concentration of mite allergens in dust from low-income homes (N = 22) with evaporative coolers in Utah County, Utah. Overall, 15 homes (68.2%) were positive for either Der p 1 or Der f 1 in at least 1 location. Geometric mean allergen levels in mattresses were 0.107 and 0.087 ug/g dust for Der p 1 and Der f 1, respectively. In furniture, levels were 0.143 and 0.165 ug/g dust for Der p1 and Der f 1, respectively. The percentage of positive homes in this study was much higher than previously reported in larger homes with swamp coolers in the same community. These results suggest socioeconomic factors may play a role in dust mite allergen prevalence in homes with evaporative coolers in dry climates.

House dust mite allergens can cause allergic diseases, including asthma, atopic dermatitis and rhinitis. Der f 20 is a novel allergen of Dermatophagoides farina (Der f), which is an arginine kinase. In the present study, the B‑cell and T‑cell epitopes of Der f 20 were predicted. The protein attribution, patterns, physicochemical properties and secondary structure of Der f 20 were also predicted. Der f 20 is a member of the ATP:guanido phosphotransferase family and contains a phosphagen kinase pattern. Using homology modeling, the present study constructed a reasonable tertiary structure of Der f 20. Using BcePred, ABCpred, BCPred and BPAP systems, B‑cell epitopes at 20‑25, 41‑49, 111‑118, 131‑141, 170‑174 and 312‑321 were predicted. Using NetMHCIIpan‑3.0 and NetMHCII‑2.2, T‑cell epitopes were predicted at 194‑202, 239‑247 and 274‑282. These results provide a theoretical basis for the design off Der f 20 epitope‑based vaccines.

House dust mite (HDM) is a predominant source of indoor aeroallergen worldwide, which induces allergic diseases including allergic rhinoconjunctivitis, allergic asthma, atopic eczema and other allergic skin diseases. Allergen specific immunotherapy (AIT) is the only potential disease-modifying treatment of HDM allergic subjects. However, AIT remains underused due to no universally accepted allergen standardization and a shortage of rigorous clinical studies to confirm safety and efficacy. With the effort of doctors and researchers in allergy field, efficacy, safety, standardization and strategy of AIT are being continuously developed. This review presents the updated research based on recently published trials and meta-analyses.

Over 89% of asthmatic children in underdeveloped countries demonstrate sensitivity to house dust mites (HDMs). The allergic response to HDMs is partially mediated by epithelial cell-derived cytokines that activate group 2 innate lymphoid cells, induce migration and activation of dendritic cells, and promote effector differentiation of HDM-specific TH2 cells. However, the contribution of innate receptor engagement on epithelial or dendritic cells by HDMs that ultimately mediates said innate and adaptive allergic responses is poorly understood. We and other investigators have demonstrated that HDMs express phosphorylcholine (PC) moieties. The major PC receptors involved in immune responses include CD36 and platelet-activating factor receptor (PAFR). Because CD36 and PAFR are expressed by epithelial cells and dendritic cells, and expression of these receptors is higher in human asthmatics, we determined whether engagement of CD36 or PAFR on epithelial or dendritic cells contributes to HDM allergy development. Testing bone marrow chimeric mice revealed that CD36 engagement on radioresistant cells and PAFR engagement on radioresistant and radiosensitive cells in the lung promote allergic responses to HDMs. Additionally, passive anti-PC IgM Abs administered intratracheally with HDMs decreased allergen uptake by epithelial cells and APCs in the lungs of C57BL/6 mice but not CD36 -/- or PAFR -/- mice. These results show that CD36 and PAFR are important mediators of HDM allergy development and that inhibiting HDM engagement with PC receptors in the lung protects against allergic airway disease., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Atopic dermatitis (AD) is a common chronic inflammatory skin disease, affecting 10-20% of individuals worldwide. Therefore, the discovery of drugs for treating AD is an attractive subject and important to human health. Diospyros kaki and Diospyros kaki (D. kaki) folium exert beneficial effects on allergic inflammation. However, the effect of D. kaki calyx on AD remains elusive. The present study evaluated the effects of an aqueous extract of D. kaki calyx (AEDKC) on AD-like skin lesions using mouse and keratinocyte models. We used a mouse AD model by the repeated skin exposure of house dust mite extract [Dermatophagoides farinae extract (DFE)] and 2,4-dinitrochlorobenzene (DNCB) to the ears. In addition, to determine the underlying mechanism of its operation, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-activated keratinocytes (HaCaT) were used. Oral administration of AEDKC decreased AD-like skin lesions, as demonstrated by the reduced ear thickness, serum immunoglobulin E (IgE), DFE-specific IgE, IgG2a, histamine level and inflammatory cell infiltration. AEDKC inhibited the expression of pro-inflammatory cytokines and a chemokine via downregulation of nuclear factor-κB and signal transducer and activator of transcription 1 in HaCaT cells. On examination of the AD-related factors in vivo and in vitro, it was confirmed that AEDKC decreased AD-like skin lesions. Taken together, the results suggest that AEDKC is a potential drug candidate for the treatment of AD.

It has always been controversial whether a single allergen performs better than multiple allergens in polysensitized patients during the allergen-specific immunotherapy. This study aimed to examine the clinical efficacy of single-allergen sublingual immunotherapy (SLIT) versus multi-allergen subcutaneous immunotherapy (SCIT) and to discover the change of the biomarker IL-4 after 1-year immunotherapy in polysensitized children aged 6-13 years with allergic rhinitis (AR) induced by house dust mites (HDMs). The AR polysensitized children (n=78) were randomly divided into two groups: SLIT group and SCIT group. Patients in the SLIT group sublingually received a single HDM extract and those in the SCIT group were subcutaneously given multiple-allergen extracts (HDM in combination with other clinically relevant allergen extracts). Before and 1 year after the allergen-specific immunotherapy (ASIT), the total nasal symptom scores (TNSS), total medication scores (TMS) and IL-4 levels in peripheral blood mononuclear cells (PBMCs) were compared respectively between the two groups. The results showed that the TNSS were greatly improved, and the TMS and IL-4 levels were significantly decreased after 1-year ASIT in both groups (SLIT group: P<0.001; SCIT group: P<0.001). There were no significant differences in any outcome measures between the two groups (for TNSS: P>0.05; for TMS: P>0.05; for IL-4 levels: P>0.05). It was concluded that the clinical efficacy of single-allergen SLIT is comparable with that of multi-allergen SCIT in 6-13-year-old children with HDM-induced AR.

House dust mite (HDM) allergy is a predominant cause for perennial allergic rhinitis (AR) in Europe. We recently reported that circulating erythrocyte numbers decrease after airway allergen challenge in a murine asthma model and in grass-pollen sensitized AR subjects. Consequently, we aimed to evaluate these findings in HDM sensitized AR subjects and the influence of preceding allergen immunotherapy. Seventy-seven (age 26.8 ± 7.3 years; 54.5% female) HDM-allergic rhinitis subjects previously enrolled in a randomized, monocentric sublingual immunotherapy (SLIT) trial at the Vienna Challenge Chamber (VCC) were included. Subjects had either received placebo (n = 22), low-dose HDM (n = 29) or high-dose HDM specific sublingual immunotherapy (n = 26) daily for 24 weeks. Blood sampling was performed before and after 6 hours of HDM allergen exposure. Overall, specific airway allergen challenge resulted in a significant decrease in circulating erythrocytes and hematocrit (p < 0.001), and elevation of leukocytes (p < 0.001), particularly segmented neutrophils (p < 0.001). Gender had no significant effect on the observed changes in circulating blood cells. Erythrocytes decreased and neutrophil counts increased significantly after airway allergen challenge regardless of preceding immunotherapy. These findings imply a rapid systemic mobilization of neutrophils occurring within immediate type hypersensitivity response upon a specific allergen challenge, which is possibly inversely linked with the erythrocyte numbers.

In the United States, the Center for Biologics Evaluation and Research of the US Food and Drug Administration regulates biologics used for diagnosis and treatment of allergic diseases. The Code of Federal Regulations 21CFR680.3(e) states that when measured, the potency of an allergenic extract is assessed according to its allergenic activity. As of 2016, 19 allergenic extracts are standardized for potency in the United States. While these standardized extracts constitute a minority of those available, they treat the most prevalent allergies (e.g. grass and ragweed pollens, dust mites, and cat) and those that induce life-threatening anaphylaxis (e.g. Hymenoptera venom). Standardization for potency enhances safety and efficacy of immunotherapy by minimizing the risks of variations in allergen dosing when switching from one lot of manufactured extract to another, and by providing an objective measure of stability of each lot of allergenic extract over time. Allergenic extracts that have multiple immunodominant allergenic proteins are standardized with little or no information about compositional differences among extracts. Here, we propose application of mass spectrometry towards measurement of compositional differences among extracts that may affect the efficacy and safety of allergen immunotherapy. In addition, we discuss of house dust mite allergen extracts as a prototypical complex extract that may be standardized by mass spectrometry., (© 2017 John Wiley & Sons Ltd.)

The environmentally widespread polysaccharide chitin is degraded and recycled by ubiquitous bacterial and fungal chitinases. Although vertebrates express active chitinases from evolutionarily conserved loci, their role in mammalian physiology is unclear. We show that distinct lung epithelial cells secrete acidic mammalian chitinase (AMCase), which is required for airway chitinase activity. AMCase-deficient mice exhibit premature morbidity and mortality, concomitant with accumulation of environmentally derived chitin polymers in the airways and expression of pro-fibrotic cytokines. Over time, these mice develop spontaneous pulmonary fibrosis, which is ameliorated by restoration of lung chitinase activity by genetic or therapeutic approaches. AMCase-deficient epithelial cells express fibrosis-associated gene sets linked with cell stress pathways. Mice with lung fibrosis due to telomere dysfunction and humans with interstitial lung disease also accumulate excess chitin polymers in their airways. These data suggest that altered chitin clearance could exacerbate fibrogenic pathways in the setting of lung diseases characterized by epithelial cell dysfunction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Allergic asthma is a heterogeneous inflammatory lung disease affecting millions of people worldwide and with a steadily increasing incidence. Mouse models have been of utmost importance in uncovering key inflammatory cell types, cytokines, and pathways in the development and maintenance of allergic asthma. Historically, the mainstay in experimental asthma research was sensitizing rodents to the model protein antigen ovalbumin (OVA) with the pro-Th2 adjuvant aluminum hydroxide, followed by repetitive OVA exposures to the airways to initiate a Th2-skewed adaptive immune response leading to eosinophilic airway inflammation and airway hyperreactivity (AHR). In the last 5 years, OVA is often replaced by naturally occurring allergens such as house dust mite (HDM) or cockroach extracts, but the principle of first sensitizing and then repetitively challenging mice with the same antigen is unchanged. Here, we describe an often used and relevant HDM-based protocol to establish acute allergic asthma, and the methods we have developed to rapidly analyze inflammatory cell infiltration in the bronchalveolar lavage fluid by flow cytometry. Moreover, we explain the methods to restimulate T cells from lung-draining mediastinal lymph nodes with HDM to allow the measurement of cytokine secretion profiles of allergen reactive T cells.

Background: Sublingual immunotherapy (SLIT) has received attention as a method for allergen immunotherapy. However, the mechanism of SLIT has not yet been fully investigated. Therefore, we evaluated the effects of SLIT in a murine asthma model, sensitized by intranasal administration of house dust mite (HDM) extracts., Methods: Female BALB/c mice were intranasally exposed to HDM for either 3 or 5 weeks (5 consecutive days per week). Mice were administered either low-dose (0.5 mg/day) or high-dose (5 mg/day) sublingual HDM extracts for 2 weeks, followed by an additional week of intranasal exposure. Airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, cytokine levels in the BALF and lymph node cell culture supernatants, and allergen-specific antibodies were measured. Lung histology was also investigated., Results: In mice sensitized for 5 weeks, high-dose SLIT ameliorated AHR, airway eosinophilia and goblet cell metaplasia. In mice sensitized for 3 weeks, even low dose SLIT ameliorated AHR and airway eosinophilia. Th2 cytokine levels in culture supernatants of submandibular lymph node cells in high-dose SLIT mice decreased, whereas IL-10 levels increased. Total IgA in BALF increased in mice sensitized for 3 or 5 weeks, and high-dose SLIT also increased allergen-specific IgG2a in mice sensitized for 5 weeks., Conclusions: These data suggest that earlier induction of SLIT in HDM-sensitized mice provides superior suppression of AHR and goblet cell metaplasia. The modulation of allergen specific IgG2a and local IgA might play a role in the amelioration of AHR and airway inflammation., (Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Allergic asthma, caused by inhaled allergens such as house dust mite or grass pollen, is characterized by reversible airway obstruction, associated with an eosinophilic inflammation of the airways, as well as airway hyper responsiveness and remodeling. The inhaled allergens trigger a type-2 inflammatory response with involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high production of immunoglobulin E (IgE) antibodies. Consequently, renewed allergen exposure results in a classic allergic response with a distinct early and late phase, both resulting in bronchoconstriction and shortness of breath. Allergen specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma and both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have proven clinical efficacy in long term suppression of the allergic response. Although these treatments are very successful for rhinitis, application of AIT in asthma is hampered by variable efficacy, long duration of treatment, and the risk of severe side-effects. A more profound understanding of the mechanisms by which AIT achieves tolerance to allergens in sensitized individuals is needed to improve its efficacy. Mouse models have been very valuable as a preclinical model to characterize the mechanisms of desensitization in AIT and to evaluate novel approaches for improved efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen absorbed to aluminum hydroxide to induce allergic sensitization, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of the allergen as immunotherapy treatment. Finally, mice are challenged by three intranasal allergen administrations. We will describe the protocols as well as the most important read-out parameters including measurement of invasive lung function measurements, serum immunoglobulin levels, isolation of broncho-alveolar lavage fluid (BALF), and preparation of cytospins. Moreover, we describe how to restimulate lung single cell suspensions, perform flow cytometry measurements to identify populations of relevant immune cells, and perform ELISAs and Luminex assays to measure the cytokine concentrations in BALF and lung tissue.

Allergic asthma is a chronic inflammatory lung disease mediated by type 2 cytokines produced by T helper 2 (Th2) cells as well as the recently discovered group 2 innate lymphoid cells (ILC2). Due to a lack of unique markers, the accurate phenotypic characterization and quantification of ILC2 requires a comprehensive panel of fluorescently labeled antibodies. The markers that are currently used to characterize ILC2 have not been standardized and often vary between research groups, which poses significant challenges when comparing data. Intranasal administration of the pro-inflammatory cytokine IL-33 in mice is associated with strong, Th2 cell-independent ILC2 activation. ILC2 are also activated in mouse models of allergic asthma based on the physiologically relevant house dust mite (HDM) allergen, which parallel eosinophilic airway inflammation observed in asthma patients. Here, we describe the analysis of ILC2 by flow cytometry in these two commonly used allergic airway inflammation models in the mouse.

Background: Protein antigens are degraded by endosomal protease in antigen presentation cell. T cells recognize peptides derived from antigen proteins bound to class II major histocompatibility complex molecules. We previously reported that an increase in the conformational stability of an antigen depressed its immunogenicity. However, there is little information on antigens with differences in molecular properties such as net charges and molecular weight., Methods: Denaturation experiments against guanidine hydrochloride. The serum IgE levels to protein antigens at 35days after the first immunization analyzed using ELISA., Results: The Der p 2 mutations in which Ile13 is mutated to Ala (I13A) and Ala122 is mutated to Ile (A122I) were shown to have lower and higher conformational stability than the wild-type, respectively, by denaturation experiments. The amount of IgE production by the less stable I13A mutant was higher and that of the stable A122I mutant was lower than that of the wild-type., Conclusion: Our results suggest that the increased conformational stability of Der p 2 depressed the IgE production in mice., General Significance: These findings should provide a milestone for the engineering of allergen vaccines., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated., Objective: To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level., Methods: Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments., Results: Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients., Conclusion: Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy., Competing Interests: Rudolf Valenta has served as a consultant for Biomay AG, Thermo Fisher Scientific, and Fresenius Medical Care and has received research grants from these companies. The other authors have no conflict of interest to declare. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Background: Interleukin-33 (IL-33) activates group 2 innate lymphoid cells (ILC2), resulting in T-helper-2 inflammation in bronchial asthma. Airway epithelial cells were reported as sources of IL-33 during apoptosis and necrosis. However, IL-33 is known to be from sources other than airway epithelial cells such as leukocytes, and the mechanisms of IL-33 production and release are not fully understood. The aim of this study was to clarify the role of IL-33 production by monocytes in airway inflammation., Methods: BALB/c mice were sensitized and challenged with a house dust mite (HDM) preparation. Airway inflammation was assessed by quantifying inflammatory cells in bronchoalveolar lavage (BAL) fluid, and IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) levels in lung. Immunohistochemistry for IL-33 in lung sections was also performed. Ly6c, CD11b, and CD11c expression was examined by flow cytometry. Clodronate liposomes were used in the HDM-airway inflammation model to deplete circulating monocytes., Results: The IL-33, but not IL-25 or TSLP, level in lung homogenates was markedly increased in HDM mice compared to control mice. IL-33-positive cells in the lungs were identified using immunohistochemistry and were increased in areas surrounding bronchi and vasculature. Furthermore, IL-33 levels were increased in mononuclear cells derived from lungs of HDM mice compared to controls. The expression of Ly6c in mononuclear cells was significantly higher in HDM mice than in controls. Treatment with clodronate liposomes led to inhibition of not only inflammatory cells in BAL fluid, airway hyper reactivity and Th2 cytokines in lung, but also IL-33 in lung., Conclusion: IL-33 from monocytes recruited to the lung may contribute to the pathogenesis of HDM-induced airway inflammation.

Allergic asthma is a chronic inflammatory disease of the conducting airways characterized by the presence of allergen-specific IgE, Th2 cytokine production, eosinophilic airway inflammation, bronchial hyperreactivity, mucus overproduction, and structural changes in the airways. Investigators have tried to mimic these features of human allergic asthma in murine models. Whereas the surrogate allergen ovalbumin has been extremely valuable for unravelling underlying mechanisms of the disease, murine asthma models depend nowadays on naturally occurring allergens, such as house dust mite (HDM), cockroach, and Alternaria alternata. Here we describe a physiologically relevant model of acute allergic asthma based on sensitization and challenge with HDM extracts, and compare it with the ovalbumin/alum-induced asthma model. Moreover, we propose a detailed readout of the asthma phenotype, determining the degree of eosinophilia in bronchoalveolar lavage fluids by flow cytometry, visualizing goblet cell metaplasia, and measuring Th cytokine production by lung-draining mediastinal lymph node cells restimulated with HDM. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

ES-62 is a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae that protects against ovalbumin (OVA)-induced airway hyper-responsiveness in mice by virtue of covalently attached anti-inflammatory phosphorylcholine (PC) residues. We have recently generated a library of small molecule analogues (SMAs) of ES-62 based around its active PC moiety as a starting point in novel drug development for asthma and identified two compounds - termed 11a and 12b - that mirror ES-62's protective effects. In this study, we have moved away from OVA, a model allergen, to test the SMAs against two clinically relevant allergens - house dust mite (HDM) and cockroach allergen (CR) extract. We show that both SMAs offer some protection against development of lung allergic responses to CR, in particular reducing eosinophil infiltration, whereas only SMA 12b is effective in protecting against eosinophil-dependent HDM-induced allergy. These data therefore suggest that helminth molecule-induced protection against model allergens may not necessarily translate to clinically relevant allergens. Nevertheless, in this study, we have managed to demonstrate that it is possible to produce synthetic drug-like molecules based on a parasitic worm product that show therapeutic potential with respect to asthma resulting from known triggers in humans., (© 2016 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1) is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD). This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family) and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase) comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL) at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBDCHIT1) structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBDCHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBDCHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBDCHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain.

Objective: To discuss the relation between the dust mite allergen (Der) in air-conditioner filters and the asthma attack., Methods: The dust samples were collected from the filters of air-conditioners in dining rooms, shopping malls, hotels and households, respectively. The concentrations of Der f 1 and Der p1 were detected by ELISA, and the dust mite immune activities were determined by dot-ELISA., Results: The concentrations of Der f 1 in the dining rooms, shopping malls, hotels and households were 1.52, 1.24, 1.31 µg/g and 1.46 µg/g respectively, and the concentrations of Der p 1 were 1.23, 1.12, 1.16 µg/ g and 1.18 µg, respectively. One hour after the running of air-conditioners, the concentrations of Der f 1 and Der p 1 in the air were higher than those before the running of air-conditioners, and the differences were significant (P < 0.05). Additionally, the dot-ELISA results revealed that the allergen extracted from the dust was capable of reacting with IgE from the sera of asthma mice allergic to dust mites., Conclusion: Der f 1 and Der p 1 appear abundantly in the filters of air-conditioners in domestic houses in Wuhu City, and the allergens can induce asthma.

Background: Modifiers of symptom severity in patients with allergic rhinoconjunctivitis (AR) are imprecisely characterized. The hygiene hypothesis implicates childhood microbial exposure as a protective factor. Cockroach sensitization (C+) might be a proxy for microbial exposure., Objective: We sought to determine whether C+ assayed by means of skin prick tests influenced AR symptom severity in controlled and natural settings., Methods: Total symptom scores (TSSs) were recorded by 21 participants with house dust mite allergy (M+) in the natural setting and during repeated exposures of 3 hours per day to house dust mite allergen in an allergen challenge chamber (ACC). In M+ participants the peripheral blood and nasal cells were assayed for T-cell activation and transcriptomic profiles (by using RNA sequencing), respectively. Participants allergic to mountain cedar (n = 21), oak (n = 34), and ragweed (n = 23) recorded TSSs during separate out-of-season exposures to these pollens (any pollen sensitization [P+]) in the ACC; a subset recorded TSSs in the pollination seasons., Results: The hierarchy of TSSs (highest to lowest) among M+ participants tracked the following skin prick test sensitization statuses: M+P+C- > M+P+C+ > M+P-C- > M+P-C+. In nasal cells and peripheral blood the immune/inflammatory responses were rapidly resolved in M+P+C+ compared with M+P+C- participants. Among those allergic to pollen, C+ was associated with a lower TSS during pollen challenges and the pollination season. After aggregated analysis of all 4 ACC studies, C+ status was associated with a 2.8-fold greater likelihood of a lower TSS compared with C- status (odds ratio, 2.78; 95% CI, 1.18-6.67; P = .02)., Conclusions: C+ status is associated with mitigation of AR symptom severity in adults with AR., (Published by Elsevier Inc.)