Non-atopic Controls Research Articles

Aspergillus fumigatus induction of IL-33 expression in chronic rhinosinusitis is PAR2-dependent.

In the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP), Aspergillus fumigatus (A. fumigatus) can upregulate IL-33 from human sinonasal epithelial cells (SNECs), which then activates innate lymphoid cells causing release of IL-13, an important driver of allergic inflammation. However, the mechanism by which A. fumigatus mediates the induction of IL-33 expression remains to be elucidated. The objectives of this study were to determine the specific fungal component(s) and the receptor responsible for mediating the A. fumigatus induced increase in IL-33 expression in SNECs from patients with CRSwNP. SNECs from CRSwNP patients were cultured and stimulated with various fungal components in the absence or presence of 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, an irreversible serine protease inhibitor, or GB83, a reversible protease activated receptor 2 (PAR2) inhibitor. IL-33 expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). PAR2 expression was examined in inflamed mucosa from nonatopic control and CRSwNP patients. Elevation of IL-33 expression in primary SNECs was found in response to fungal protease but not fungal cell wall components. PAR2 expression was elevated in inflamed mucosa from CRSwNP patients in comparison to controls. The A. fumigatus fungal protease-mediated elevation in IL-33 expression by human SNECs was serine protease- and PAR2-dependent. These data suggest that serine protease activity of A. fumigatus is capable of inducing IL-33 expression in CRSwNP SNECs via PAR2, a potential therapeutic target in the treatment of CRSwNP. NA Laryngoscope, 129:2230-2235, 2019.

Development of a progesterone-specific IgE assay for diagnosing patients with suspected progestogen hypersensitivity

BackgroundProgesterone hypersensitivity (PH) manifests as a spectrum of allergic symptoms during the luteal phase of the menstrual cycle. Confirming progesterone-specific immunoglobulin E (IgE; sIgE) by skin testing is unreliable because of irritant responses. ObjectiveTo develop a progesterone sIgE assay to assist in diagnosing PH. MethodsA progesterone–bovine serum albumin (BSA) conjugate was characterized and used to analyze sera collected from women in our center with suspected PH in a 1-batch enzyme-linked immunosorbent assay (ELISA) to establish high, low and negative cut-points. Sera collected from healthy nonatopic female subjects and from women with classical PH symptoms were included as negative and positive controls, respectively. Values exceeding the average negative control (OD + 3× the standard deviation) were considered positive. These cut-points were subsequently used to establish positive and negative results for serum from women with suspected PH received from other centers. A subset of high positive sera was used for ELISA-inhibition and in a beta-hexosaminidase mediator release assay to evaluate the specificity and functional relevance of progesterone-specific serum IgE, respectively. The numbers of true negative, false negative, true positive, and false positive samples were determined. ResultsThe direct progesterone sIgE ELISA results ranged from high positive to low positive and negative compared with healthy nonatopic control sera. Enzyme-linked immunosorbent assay inhibition and beta-hexosaminidase mediator release confirmed specificity and functional relevance of progesterone-sIgE, respectively. The sensitivity, specificity positive predictive values and negative predictive values were found to be 82%, 100%, 86%, and 100%, respectively, using the mediator release assay results as the gold standard. ConclusionThis assay has a good specificity and positive predictive value for screening women with suspected PH for progesterone sIgE.

Genetic determinants of paediatric food allergy: A systematic review.

The genetic determinants of food allergy have not been systematically reviewed. We therefore systematically reviewed the literature on the genetic basis of food allergy, identifying areas for further investigation. We searched three electronic databases (MEDLINE, EMBASE and PubMed) on 9 January 2018. Two authors screened retrieved articles for review according to inclusion criteria and extracted relevant information on study characteristics and measures of association. Eligible studies included those that reported an unaffected nonatopic control group, had genetic information and were carried out in children. Of the 2088 studies retrieved, 32 met our inclusion criteria. Five were genome-wide association studies, and the remaining were candidate gene studies. Twenty-two of the studies were carried out in a predominantly Caucasian population with the remaining 10 from Asian-specific populations or unspecified ethnicity. We found FLG, HLA, IL10, IL13, as well as some evidence for other variants (SPINK5, SERPINB and C11orf30) that are associated with food allergy. Little genetic research has been carried out in food allergy, with FLG, HLA and IL13 being the most reproducible genes for an association with food allergy. Despite promising results, existing genetic studies on food allergy are inundated with issues such as inadequate sample size and absence of multiple testing correction. Few included replication analyses or population stratification measures. Studies addressing these limitations along with functional studies are therefore needed to unravel the mechanisms of action of the identified genes.

Evidence for a Role of TGF-β-Activated Kinase 1 and MAP3K7 Binding Protein 3 in Peanut-Specific T-Cell Responses

Peanut allergy is considered to be the most common cause for food-induced anaphylaxis. Currently, no approved treatment is available. Avoidance is the only measure to prevent anaphylactic reactions to peanuts. T-helper cells are of special importance for the sensitization process and the maintenance of allergic inflammation. Identifying markers of allergen-specific T-cell responses may help to develop novel treatment approaches. Therefore, we aimed to define new T-cell target genes in Ara h 2-specific T cells and to investigate the possibility of using them as biomarkers of peanut allergy in peripheral blood mononuclear cells (PBMCs). We performed whole mRNA array analysis (whole human genome oligo microarray) of in vitro expanded Ara h 2-specific T cells (CFSE^lowCD3⁺CD4⁺) from 5 peanut-allergic (PA) and 5 non-peanut-sensitized individuals. Expression of selected genes as a result of a two-step bioinformatic approach was confirmed in a second cohort by quantitative PCR. TGF-β- activated kinase 1 and MAP3K7 binding protein 3 (TAB3), calcium/calmodulin-dependent protein kinase type IV (CAMK4) and HemK methyltransferase family member 1 (HEMK1) were significantly upregulated in Ara h 2-specific T cells of PA patients. In addition, the expression of these genes was also assessed in unstimulated PBMCs from a cohort (n = 43) of PA, atopic non-PA, and nonatopic controls. Interestingly, in unstimulated PBMCs, TAB3 expression was significantly downregulated in PA patients compared to atopic non-PA individuals. Thus, TAB3 may play a significant role at the level of T-cell activation and may also be a candidate biomarker for PA.

Differentiation between control subjects and patients with chronic spontaneous urticaria based on the ability of anti-IgE autoantibodies (AAbs) to induce FcεRI crosslinking, as compared to anti-FcεRIα AAbs

BackgroundThe reported prevalences of IgG autoantibodies (AAbs) to FcεRIα and IgE in sera from patients with chronic spontaneous urticaria (CSU) have varied, and these AAbs are also often observed in healthy control subjects. Regarding the histamine release activity of purified IgG from patients with CSU, the number of examined patients has been small. Thus, we sought to determine the prevalence and FcεRI crosslinking ability of these AAbs in a large number of patients with CSU and non-atopic control (NC) subjects. MethodsWe compared the concentrations of anti-IgE and anti-FcεRIα AAbs and the abilities of these AAbs to cause FcεRI aggregation in patients with CSU (n = 134) and NC subjects (n = 55) using ELISA and an in vitro elicitation test, respectively. ResultsThe concentration of anti-IgE AAbs was significantly different between the NC subjects and the CSU patients (P < 0.0001, cutoff value: 0.558 μg/mL), whereas the concentration of anti-FcεRIα AAbs was not. A significant difference in the duration of illness was noted between patients with lower and those with higher concentrations of anti-IgE AAbs relative to the cutoff value. The abilities of anti-IgE AAbs, but not anti-FcεRIα AAbs, to induce FcεRI crosslinking were significantly higher in CSU patients than in NC subjects (P = 0.0106). ConclusionsIn the Japanese population of CSU patients studied, the ability of the anti-IgE AAbs to induce FcεRI crosslinking differed significantly between NC subjects and CSU patients, suggesting the involvement of anti-IgE AAbs in the pathogenesis of CSU in the Japanese population.

The nonlesional skin surface distinguishes atopic dermatitis with food allergy as a unique endotype.

Skin barrier dysfunction has been reported in both atopic dermatitis (AD) and food allergy (FA). However, only one-third of patients with AD have FA. The purpose of this study was to use a minimally invasive skin tape strip sampling method and a multiomics approach to determine whether children with AD and FA (AD FA+) have stratum corneum (SC) abnormalities that distinguish them from AD without FA (AD FA-) and nonatopic (NA) controls. Transepidermal water loss was found to be increased in AD FA+. Filaggrin and the proportion of ω-hydroxy fatty acid sphingosine ceramide content in nonlesional skin of children with AD FA+ were substantially lower than in AD FA- and NA skin. These abnormalities correlated with morphologic changes in epidermal lamellar bilayer architecture responsible for barrier homeostasis. Shotgun metagenomic studies revealed that the nonlesional skin of AD FA+ had increased abundance of Staphylococcus aureus compared to NA. Increased expression of keratins 5, 14, and 16 indicative of hyperproliferative keratinocytes was observed in the SC of AD FA+. The skin transcriptome of AD FA+ had increased gene expression for dendritic cells and type 2 immune pathways. A network analysis revealed keratins 5, 14, and 16 were positively correlated with AD FA+, whereas filaggrin breakdown products were negatively correlated with AD FA+. These data suggest that the most superficial compartment of nonlesional skin in AD FA+ has unique properties associated with an immature skin barrier and type 2 immune activation.

Serum Proteome Profiling to Identify Proteins Promoting Pathogenesis of Non-atopic Asthma.

Asthma is the inflammatory disorder of airways highly prevalent in both, children and adults all over the world. The aim of this study was to investigate the serum proteome for the identification of proteins contributing to the pathogenesis of non-atopic asthma. Protein expression profiling of sera from non-atopic asthmatic patients (n=73) and controls (n=99) was carried out using 1D SDS PAGE. Differentially expressed protein bands were compared with controls, cut from the gel and identified by LC-Q-TOF_MSMS analysis. Various acute phase proteins (i.e., haptoglobin, transthyretin, serum amyloid A, and serum albumin), retinol binding protein 4 (RBP4), Ig gamma-1-chain C-region, hemoglobin subunit β, complement system components (C8 gamma chain and C4-A), ApoA-1 and Ig κ chain C-region containing protein fractions were predominantly down-regulated in asthmatic patients. Similarly, a higher proportion of male patients exhibited down-regulation of all other (except albumin and C4-A containing) protein fractions as compared to female patients. However, fractions consisting of another acute phase protein, inter-alpha-trypsin heavy chain 4 (ITIH4) and complement system component C3 were up-regulated in the majority of patients. Our study is the first to confirm the role of thyroxine (TTR), retinol (RBP4), cholesterol (apoA-1) transporting proteins, inflammation and its mediators in the pathogenesis of asthma. Further study may help to improve diagnosis and plan better treatment strategies.

Quercetin inhibits Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R related Ca2+ fluctuations

An allergic reaction is a potentially fatal hypersensitivity response caused by mast cell activation, particularly histamine and lipid mediators. Histamine release caused by reaction to drugs is considered a pseudo-allergic reaction. Quercetin is known for its anti-allergic immune effect. However, at present, its anti-pseudo-allergic effect and its mechanism are less investigated. Therefore, the purpose of this study was to evaluate the anti-pseudo-allergic effect of Quercetin in vivo and to explore the mechanism in vitro. The anti-pseudo-allergic activity of Quercetin was evaluated in vivo using a mouse model, while Quercetin mechanism of action was examined in vitro using HEK293 cells expressing Mrgprx2, a mast cell specific receptor, and LAD2 mast cell line. Our in vivo results showed that Quercetin could attenuate Evans blue leakage in the paws and hind paw thickness in C57BL/6 mice in a dose-dependent manner, and could significantly inhibit serum histamine and chemokines release. In addition, it suppressed calcium mobilization and attenuated the release of histamine and MCP-1 in peritoneal mast cells in a dose-dependent manner. Furthermore, it inhibited the vasodilation due to histamine, the release of eosinophils, and the percentage of degranulated mast cells, indicating that Quercetin antagonized mast cell mediators in vivo, histamine-induced vasodilation and eosinophil release.In vitro results showed that Quercetin reduced pseudo-allergic induced calcium influx, suppressed degranulation and chemokines release in a similar way as dexamethasone (100 μM) (mast cell stabilizer) in LAD2 mast cell line. In addition, Quercetin inhibited Mrgprx2-induced both calcium influx and pseudo-allergic reaction in HEK293 cells expressing Mrgprx2. C48/80, a histamine promoter, and Substance P (a neuropeptide) EC50 was higher when combined with Quercetin compared to the EC50 of these compounds alone, suggesting that Quercetin could inhibit Mrgprx2-induced pseudo-allergic reaction. Furthermore, Quercetin decreased PLCγ-IP3R signaling pathway activation induced by C48/80 in LAD2 mast cell line. In Mrgprx2 knockdown LAD2 cells, the effect of Quercetin (200 μM) reduced C48/80 induced calcium flux and the release of β‑hexosaminidase, histamine, MCP-1 and IL-8 compared with non-atopic control (NC) transfected LAD2 human mast cells, suggesting that Quercetin anti-pseudo-allergic effect was related to Mrgprx2. The docking results showed that Quercetin had a good binding affinity with Mrgprx2 similar to the one of Substance P and C48/80. Therefore, Quercetin inhibited Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations.Our results validated Quercetin as an effective small molecule inhibiting Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations, thus highlighting a potential candidate to suppress Mrgprx2 induced pseudo-allergic related diseases.

Markers of atopic dermatitis, allergic rhinitis and bronchial asthma in pediatric patients: correlation with filaggrin, eosinophil major basic protein and immunoglobulin E

BackgroundAllergic reactions have been implicated as contributions in a number of atopic disorders, including atopic dermatitis (AD), allergic rhinitis (AR) and bronchial asthma (BA). However, the potential for filaggrin protein, eosinophil major basic protein (MBP) and immunoglobulin E (IgE) to elicit allergic response or to contribute to atopic disorders remains largely unexplored in pediatric patients. This study was undertaken to investigate the status and contribution of filaggrin protein, eosinophil MBP and total IgE in pediatric patients with AD, AR and BA.MethodsSera from 395 pediatric patients of AD, AR or BA with varying levels of disease activity according to the disease activity index and 410 age-matched non-atopic healthy controls were evaluated for serum levels of atopic markers, including filaggrin, eosinophil MBP and IgE.ResultsSerum analysis showed that filaggrin levels were remarkably high in pediatric patients with AD, followed by BA and AR, whereas its levels were low in non-atopic pediatric controls. Eosinophil MBP levels in sera of atopic patients were significantly high as compared with their respective controls, but its levels were highest in AR patients, followed by AD and BA. Total IgE in sera of AD patients was markedly high, followed by AR and BA patients, whereas its levels were low in non-atopic pediatric controls. Interestingly, not only was an increased number of subjects positive for filaggrin protein, eosinophil MBP or total IgE, but also their levels were statistically significantly higher among those atopic patients whose disease activity scores were higher as compared with atopic patients with lower disease activity scores.ConclusionsThese findings strongly support a role of filaggrin protein, eosinophil MBP and IgE in the onset of allergic reactions in pediatric patients with AD, AR and BA. The data suggest that filaggrin, eosinophil MBP or IgE might be useful in evaluating the progression of AD, AR or BA and in elucidating the mechanisms involved in the pathogenesis of these pediatric disorders.

Enterovirus infection during pregnancy is inversely associated with atopic disease in the offspring.

Prenatal environment has been shown to influence child's risk of atopic diseases. Laboratory-confirmed data about the role of maternal infections during pregnancy is scarce. The aim of this study was to determine the associations between serologically confirmed maternal infections during pregnancy and atopic disease in the offspring. This was a nested case-control study within a prospective birth cohort study. Altogether 202 atopic case children and 333 matched non-atopic control children were included. Atopic outcome was defined as having an atopic disease and IgE sensitization by the age of 5years. We analysed serologically acute enterovirus (EV), influenza virus A (IAV) and Mycoplasma pneumoniae (M.pneumoniae) infections during pregnancy, and mother's seropositivity against human cytomegalovirus (CMV) and Helicobacter pylori. Maternal EV infection during pregnancy was inversely associated with atopic outcome in the offspring (odds ratio 0.43; 95% confidence interval: 0.23-0.80, P=0.008). Acute IAV or M.pneumoniae infections or seropositivity against CMV or Helicobacter pylori were not associated with the atopic outcome. Our results suggest that maternal EV infections during pregnancy are inversely associated with atopic disease in the offspring. Our finding provides further support to the previous studies suggesting an important role of the in utero environment in the development of atopic diseases.

Monocytes accumulate in the airways of children with fatal asthma.

Activated T helper type 2 (Th2) cells are believed to play a pivotal role in allergic airway inflammation, but which cells attract and activate Th2 cells locally have not been fully determined. Recently, it was shown in an experimental human model of allergic rhinitis (AR) that activated monocytes rapidly accumulate in the nasal mucosa after local allergen challenge, where they promote recruitment of Th2 cells and eosinophils. To investigate whether monocytes are recruited to the lungs in paediatric asthma. Tissue samples obtained from children and adolescents with fatal asthma attack (n = 12), age-matched non-atopic controls (n = 9) and allergen-challenged AR patients (n = 8) were subjected to in situ immunostaining. Monocytes, identified as CD68+S100A8/A9+ cells, were significantly increased in the lower airway mucosa and in the alveoli of fatal asthma patients compared with control individuals. Interestingly, cellular aggregates containing CD68+S100A8/A9+ monocytes obstructing the lumen of bronchioles were found in asthmatics (8 out of 12) but not in controls. Analysing tissue specimens from challenged AR patients, we confirmed that co-staining with CD68 and S100A8/A9 was a valid method to identify recently recruited monocytes. We also showed that the vast majority of accumulating monocytes both in the lungs and in the nasal mucosa expressed matrix metalloproteinase 10, suggesting that this protein may be involved in their migration within the tissue. Monocytes accumulated in the lungs of children and adolescents with fatal asthma attack. This finding strongly suggests that monocytes are directly involved in the immunopathology of asthma and that these pro-inflammatory cells are potential targets for therapy.

The immunomodulatory role of zinc in asthmatic patients

BackgroundZinc deficiency may play an important role in the development of atopic asthma. The aim of the workTo assess serum zinc levels in adult atopic, non-atopic asthmatic patients, and in healthy controls and to investigate its modulatory effect on production of interferon gamma (IFN-γ) and interleukin-10 (IL-10) by peripheral blood mononuclear cells (PBMCs) in vitro. MethodsSixty asthmatics and 30 apparently healthy volunteers were included in this study. All patients were subjected to history taking, clinical examination, pulmonary function tests, skin prick test (SPT), serum zinc assessment by a colorimetric method as well as serum total IgE measurement by Enzyme-linked immunosorbent assay (ELISA). PBMCs were activated in vitro in the presence and absence of zinc, and then cell culture supernatants were analyzed for IFN-γ and IL-10 by ELISA. ResultsSerum zinc levels were significantly lower in atopic asthmatics than non-atopic asthmatics and healthy controls. In atopic asthmatics, highly significant correlations were found between zinc levels and total Ig E levels as well as FEV1. In culture, zinc triggers IFN-γ and inhibits IL-10 production by PBMCs, in atopic asthmatics. In non atopic asthmatics and healthy controls, IFN-γ and IL-10 were slightly affected by zinc supplementation in culture. ConclusionSerum zinc levels affect asthma phenotypes. Atopic asthmatics might benefit from zinc supplements.

Interleukin-25 and eosinophils progenitor cell mobilization in allergic asthma

BackgroundEosinophil-lineage committed progenitor cells (EoP) migrate from the bone marrow and differentiate locally to provide an ongoing source of mature eosinophils in asthmatic inflammatory responses in the airways. Sputum levels of EoP are increased in asthmatics compared to normal controls suggesting an exaggerated eosinophilopoietic environment in the airways. Understanding what factors promote EoP traffic to the airways is important to understand the diathesis of asthma pathology. Interleukin (IL)-25, is an epithelial-derived cytokine that promotes type 2 inflammatory responses. We have previously shown that levels of IL-25 and expression of the IL-25 receptor (IL-17RA and IL-17RB) on mature eosinophils are greater in allergic asthmatics compared to atopic non-asthmatics and non-atopic normal controls. In addition, these levels were increased significantly increased following allergen inhalation challenge and physiologically relevant levels of IL-25 stimulated eosinophil degranulation, intracellular IL-5 and IL-13 expression and primed migration to eotaxin. The current study, examined the role of IL-25 on allergen-induced trafficking of EoP in atopic asthmatics.MethodsAsthmatics (n = 14) who developed allergen-induced early and late responses were enrolled in the study. Blood was collected at pre- and 24 h post-challenge. At each time point, surface expression of IL-17RA and IL-17RB on EoP was evaluated by flow cytometry. Migration assays examined the effect of IL-25 on EoP chemotactic responses, in vitro. In addition, IL-25 knockout ovalbumin (OVA) sensitized and challenged mice were studied to evaluate in vivo mobilization effects of IL-25 on newly formed EoP and mature eosinophils.ResultsThere was a significant increase in numbers of blood EoP expressing IL-17RB, 24 h post-allergen inhalation challenge in allergic asthmatics. Pre-exposure to IL-25 primed the migrational responsiveness of EoP to stromal cell-derived factor 1α. In OVA-sensitized mice, knocking out IL-25 significantly alleviated OVA-induced eosinophil infiltration in the airway and newly formed eosinophils were reduced in the lung.ConclusionsThe findings of this study indicate a potential role for IL-25 in allergen-induced trafficking of EoP to the airways and local differentiation promoting tissue eosiniophilia in asthmatic responses.

Increased healthcare utilisation among atopic children in a general practice database: a nested index-control study.

BackgroundAtopic eczema, asthma, and allergic rhinitis (AR) create a serious burden on general practice resources.AimTo investigate the use of general practice resources (that is, consultation visits, telephone contacts, and home visits) in children with physician-diagnosed atopic disorders (ADs).Design & settingIn a nested index-control study design, all children (here defined as individuals aged 2–18 years) listed in a representative general practice database were selected in 2014.MethodChildren diagnosed with ADs were matched on age and sex with non-atopic controls within the same practice. For all the different groups, the number and frequency of children contacting the GP were calculated.ResultsOf the children with atopic eczema (n = 15 202), 80% consulted the GP in 2014 (controls = 67%). Of the children with asthma (n = 7754), 80% consulted the GP (controls = 65%), and for children with AR (n = 6710), this was 82% (controls = 66%). Of the children with all three ADs, 91% consulted the GP (controls = 68%). On average, a child with atopic eczema contacted the GP 2.8 times/year (controls = 1.9); for children with asthma, the contact frequency was 3.0 (controls = 1.9); and for AR, 3.2 (controls = 1.9). For children with all three ADs, the contact frequency was 4.3 (controls = 2.0). Consultations related to the ADs investigated only explain a smaller part of the increased healthcare utilisation in atopic children.ConclusionAtopic children use more general practice resources compared to non-atopic children, yet frequently for morbidity or other health-related questions not related to one of the ADs.

Association of REL Polymorphism with Cow's Milk Proteins Allergy in Pediatric Algerian Population

ABSTRACTIntroduction: Cow's milk proteins allergy (CMPA) pathogenesis involves complex immunological mechanisms with the participation of several cells and molecules involved in food allergy. The association of polymorphisms in the interleukin 4, Forkhead box P3 and the avian reticuloendotheliosis genes was investigated in an infant population with CMPA of Western Algeria. Materials and methods: We obtained DNA and clinical data from milk allergic subjects during active phase and from a group of non-atopic control subjects. Results: Our findings showed that the allele G of the cRel gene intronic polymorphism at +7883 positions was significantly higher among cow's milk proteins allergic patients compared to control subjects. Conclusion: The results of this study suggest a possible association of CMPA with cRel G+7883T polymorphism.

Innate Immune Response to Viral Infections in Primary Bronchial Epithelial Cells is Modified by the Atopic Status of Asthmatic Patients.

PurposeIn order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls.MethodsHBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-β and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR.ResultsPIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-β mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-β, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-β mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics.ConclusionsThe immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status.

Human Bronchial Epithelial Cell-derived Factors from Severe Asthmatic Subjects Stimulate Eosinophil Differentiation.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

A shift in the IL-6/STAT3 signalling pathway imbalance towards the SHP2 pathway in severe asthma results in reduced proliferation process

BackgroundBronchial fibroblasts are the main structural cells responsible for extracellular matrix production and turnover in lung tissue. They play a key role in airway remodelling in asthma through different cytokines including interleukin (IL-6). ObjectiveTo decipher IL-6 signalling in bronchial fibroblasts obtained from severe eosinophilic asthmatics compared to mild asthmatics and healthy controls. MethodsHuman bronchial fibroblasts were isolated from bronchial biopsies of mild and severe eosinophilic asthmatics and non-atopic healthy controls. IL-6 was assessed by qRT-PCR and ELISA. Phosphorylated STAT3, SHP2 and p38/MAPK were evaluated by Western blot. Chemical inhibitors for SHP2 and p38 were used. Fibroblast proliferation was evaluated by BrdU incorporation test. ResultsIL-6 release was significantly increased in fibroblasts from mild and severe asthmatics compared to healthy controls. Fibroblasts from severe asthmatics showed a reduced STAT3 activation compared to mild asthmatics and healthy controls. Constitutive activation of phosphatase SHP2 was found to negatively regulate IL-6 induced STAT3 phosphorylation in fibroblasts from severe asthmatics. This effect was accompanied by a decrease in fibroblast proliferation rate due to the activated p38/mitogen-activated protein kinase. SHP2 and p38/MAPK specific inhibitors (PHPS1 and SB212190) significantly induce a restoration of STAT3 phosphorylation, IL-6 target gene expression and cell proliferation. ConclusionThese data show dysregulated IL-6 signalling in bronchial fibroblasts derived from severe eosinophilic asthmatic subjects involving the protein tyrosine phosphatase SHP2 and p38MAPK. Collectively, our data provides new insights into the mechanisms by which bronchial fibroblasts regulate airway remodelling in severe asthma.

Risks for comorbidity in children with atopic disorders: an observational study in Dutch general practices

ObjectiveThis study aimed to investigate both atopic and non-atopic comorbid symptoms and diseases in children with physician-diagnosed atopic disorders (atopic eczema, asthma and allergic rhinitis).MethodsAll children aged 0–18 years listed...

Atopic children and use of prescribed medication: A comprehensive study in general practice.

PurposeA comprehensive and representative nationwide general practice database was explored to study associations between atopic disorders and prescribed medication in children.MethodAll children aged 0–18 years listed in the NIVEL Primary Care Database in 2014 were selected. Atopic children with atopic eczema, asthma and allergic rhinitis (AR) were matched with controls (not diagnosed with any of these disorders) within the same general practice on age and gender. Logistic regression analyses were performed to study the differences in prescribed medication between both groups by calculating odds ratios (OR); 93 different medication groups were studied.ResultsA total of 45,964 children with at least one atopic disorder were identified and matched with controls. Disorder-specific prescriptions seem to reflect evidence-based medicine guidelines for atopic eczema, asthma and AR. However, these disorder-specific prescriptions were also prescribed for children who were not registered as having that specific disorder. For eczema-related medication, about 3.7–8.4% of the children with non-eczematous atopic morbidity received these prescriptions, compared to 1.4–3.5% of the non-atopic children. The same pattern was observed for anti-asthmatics (having non-asthmatic atopic morbidity: 0.8–6.2% vs. controls: 0.3–2.1%) and AR-related medication (having non-AR atopic morbidity: 4.7–12.5% vs. controls: 2.8–3.1%). Also, non-atopic related medication, such as laxatives and antibiotics were more frequently prescribed for atopic children.ConclusionsThe present study shows that atopic children received more prescriptions, compared to non-atopic children. Non-atopic controls frequently received specific prescriptions for atopic disorders. This indicates that children with atopic disorders need better monitoring by their GP.