IgE Epitopes of the House Dust Mite Allergen Der p 7 Are Mainly Discontinuous and Conformational.

Background: Several studies indicate that Der p 7 is an important and clinically relevant allergen of Dermatophagoides pteronyssinus which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7. Methods: Recombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1-P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes. Results: rDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7. Conclusion and Clinical Relevance: IgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides.

High Th2 cytokine levels and upper airway inflammation in human inherited T-bet deficiency.

We have described a child suffering from Mendelian susceptibility to mycobacterial disease (MSMD) due to autosomal recessive, complete T-bet deficiency, which impairs IFN-γ production by innate and innate-like adaptive, but not mycobacterial-reactive purely adaptive, lymphocytes. Here, we explore the persistent upper airway inflammation (UAI) and blood eosinophilia of this patient. Unlike wild-type (WT) T-bet, the mutant form of T-bet from this patient did not inhibit the production of Th2 cytokines, including IL-4, IL-5, IL-9, and IL-13, when overexpressed in T helper 2 (Th2) cells. Moreover, Herpesvirus saimiri-immortalized T cells from the patient produced abnormally large amounts of Th2 cytokines, and the patient had markedly high plasma IL-5 and IL-13 concentrations. Finally, the patient's CD4+ αß T cells produced most of the Th2 cytokines in response to chronic stimulation, regardless of their antigen specificities, a phenotype reversed by the expression of WT T-bet. T-bet deficiency thus underlies the excessive production of Th2 cytokines, particularly IL-5 and IL-13, by CD4+ αß T cells, causing blood eosinophilia and UAI. The MSMD of this patient results from defective IFN-γ production by innate and innate-like adaptive lymphocytes, whereas the UAI and eosinophilia result from excessive Th2 cytokine production by adaptive CD4+ αß T lymphocytes.

GITRL on dendritic cells aggravates house dust mite-induced airway inflammation and airway hyperresponsiveness by modulating CD4+ T cell differentiation.

BACKGROUND: Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) plays an important role in tumors, autoimmunity and inflammation. However, GITRL is not known to modulate the pathogenesis of allergic asthma. In this study, we investigated whether regulating GITRL expressed on dendritic cells (DCs) can prevent asthma and to elucidate its mechanism of action. METHODS: In vivo, the role of GITRL in modulating house dust mite (HDM)-induced asthma was assessed in adeno-associated virus (AAV)-shGITRL mice. In vitro, the role of GITRL expression by DCs was evaluated in LV-shGITRL bone marrow dendritic cells (BMDCs) under HDM stimulation. And the direct effect of GITRL was observed by stimulating splenocytes with GITRL protein. The effect of regulating GITRL on CD4+ T cell differentiation was detected. Further, GITRL mRNA in the peripheral blood of asthmatic children was tested. RESULTS: GITRL was significantly increased in HDM-challenged mice. In GITRL knockdown mice, allergen-induced airway inflammation, serum total IgE levels and airway hyperresponsiveness (AHR) were reduced. In vitro, GITRL expression on BMDCs was increased after HDM stimulation. Further, knocking down GITRL on DCs partially restored the balance of Th1/Th2 and Th17/Treg cells. Moreover, GITRL stimulation in vitro inhibited Treg cell differentiation and promoted Th2 and Th17 cell differentiation. Similarly, GITRL mRNA expression was increased in the peripheral blood from asthmatic children. CONCLUSIONS: This study identified a novel role for GITRL expressed by DCs as a positive regulator of CD4+ T cells responses in asthma, which implicates that GITRL inhibitors may be a potential immunotherapy for asthma.

Obesity-induced Vitamin D Deficiency Contributes to Lung Fibrosis and Airway Hyperresponsiveness.

Vitamin D (VitD) has pleiotropic effects. VitD deficiency is closely involved with obesity and may contribute to the development of lung fibrosis and aggravation of airway hyperresponsiveness (AHR). We evaluated the causal relationship between VitD deficiency and the lung pathologies associated with obesity. In vivo effects of VitD supplementation were analyzed using high-fat diet (HFD)-induced obese mice and TGF-ß1 (transforming growth factor-ß1) triple transgenic mice. Effects of VitD supplementation were also evaluated in both BEAS-2B and primary lung cells from the transgenic mice. Obese mice had decreased 25-OH VitD and VitD receptor expressions with increases of insulin resistance, renin and angiotensin-2 system (RAS) activity, and leptin. In addition, lung pathologies such as a modest increase in macrophages, enhanced TGF-ß1, IL-1ß, and IL-6 expression, lung fibrosis, and AHR were found. VitD supplementation to HFD-induced obese mice recovered these findings. TGF-ß1-overexpressing transgenic mice enhanced macrophages in BAL fluid, lung expression of RAS, epithelial-mesenchymal transition markers, AHR, and lung fibrosis. VitD supplementation also attenuated these findings in addition to the attenuation of the expressions of TGF-ß1, and phosphorylated Smad-2/3 in lung. Supplementing in vitro-stimulated BEAS-2B and primary lung cells with VitD inhibited TGF-ß1 expression, supporting the suppressive effect of VitD for TGF-ß1 expression. These results suggest that obesity leads to VitD deficiency and worsens insulin resistance while enhancing the expression of leptin, RAS, TGF-ß1, and proinflammatory cytokines. These changes may contribute to the development of lung fibrosis and AHR. VitD supplementation rescues these changes and may have therapeutic potential for asthma with obesity.

In utero exposure to genistein decreased intranasal house dust mite-induced respiratory allergy in middle-aged male B6C3F1 offspring.

Despite many hypothesized benefits of dietary isoflavone genistein (GEN) deriving from soy-based products, questions surrounding GEN's developmental effects are increasing. To understand if in utero GEN exposure modulated postnatal respiratory allergies in the middle age, we conducted a time course study in the B6C3F1 offspring (PND 240-330) using a common household allergen (house dust mites: HDM; 10 µg/mouse for PND 240 and 290, and 50 µg/mouse for PND 330, a middle age in mice) following intranasal instillation, a physiological route of allergen exposure. GEN was administered to dams by gavage from gestational day 14 to parturition at a physiologically relevant dose (20 mg/kg body weight). Female and male offspring were sensitized with HDM allergens beginning about one month prior to sacrifice followed by challenges with three weekly dosings of HDM extracts, and they were euthanized at day 3 following the final HDM exposure. In utero exposure to GEN decreased HDM allergen-induced respiratory allergy in male B6C3F1 offspring at PND 330 as reflected by decreases in airway hyperresponsiveness (e.g., Penh value), HDM-specific IgG1 (a Th2 type Ab) and the activity of eosinophil peroxidase in the lung (an indication of eosinophil recruitment to the lungs). However, in utero exposure to GEN had minimal effects on HDM allergen-induced respiratory allergy in the middle-aged female offspring. Changes in serum total IgE, HDM-specific IgE, and lung histopathology scores in both male and female offspring were not biologically significant. Overall, in utero GEN exposure exerted a protective effect on respiratory allergy in the middle-aged male, but not female, B6C3F1 offspring following later-life HDM exposures.

Occupational respiratory allergy to lettuce in lettuce farmers.

BACKGROUND: Lettuce-associated respiratory allergy has never been reported before. The aim of this study was to clarify the clinical condition of lettuce-associated respiratory allergy and to identify the lettuce antigen which induces allergic symptoms. METHODS: We distributed questionnaires to 1168 lettuce farmers and performed medical examinations in those who exhibited respiratory symptoms related to occupational exposure to lettuce. We analysed specific IgE-binding proteins in the sera of patients through immunoblotting analysis and determined molecular characterization of the IgE-binding bands using liquid chromatography-mass spectrometry. RESULTS: A total of 932 farmers (80%) responded to the questionnaire. Of those, 7% exhibited lettuce-associated respiratory symptoms, during harvesting and packaging. Thirteen patients were diagnosed with allergy to lettuce and agreed to undergo further examinations. The percentage of activated basophils in these patients was significantly higher compared with that reported in negative controls (P < .05). Lettuce-specific IgE (ImmunoCAP® ) and skin prick testing was positive in 46% and 62% of patients, respectively. Notably, occupational lettuce-allergic asthma was detected in one patient through specific bronchial provocation testing. The IgE-binding bands recognized in the sera of >50% of patients were identified as epidermis-specific secreted glycoprotein EP1-like (51 kDa). CONCLUSION: The present analysis identified a novel lettuce allergen. This allergen may have clinically useful applications, such as specific IgE testing and allergen-specific immunotherapy.

Trends in aeroallergen sensitization in Germany - An analysis of 2919 serological data sets of a university ENT department.

Background: Recently, population-based birth-cohort studies provided an insight into the allergic march during childhood.Aims: Our study aimed to investigate sensitization pattern until advanced age.Patients and methods: Demographic, clinical and serological characteristics of 2919 patients with positive allergen-specific IgE between 1999 and 2019 were analyzed. We performed subgroup analysis of various age-groups and different years of birth to distinguish between age-dependent changes and birth-cohort-effects.Results: Since 1999, the proportion of sensitized children has significantly increased. The prevalence of sIgE towards most allergens reached its peak in adolescence or young adulthood. Only to mites, the highest rate of sensitization was found in childhood. With further aging, the prevalence of sIgE significantly decreased in most sensitizations. Only to Fagales, the highest rate of sensitization was observed among patients >65 years. The year-of-birth analysis proved the above-mentioned changes to be age-dependent. Further, it revealed various sensitization trends from older to younger generations.Conclusions and significance: The increased proportion of children with sensitization during the last 20 years outlines the allergy epidemic. Probably due to immunosenescence, the aeroallergen sensitization rates decreased with aging, except for Fagales. Over time, different aeroallergens gained or lost relevance, potentially due to environmental and life-style changes.

Physiological responses to cisplatin using a mouse hypersensitivity model.

Background: The physiological mechanisms underlying the development of respiratory hypersensitivity to cisplatin (CDDP) are not well-understood. It has been suggested that these reactions are likely the result of type I hypersensitivity, but other explanations are plausible and the potential for CDDP to induce type I hypersensitivity responses has not been directly evaluated in an animal model. Objectives and Methods: To investigate CDDP hypersensitivity, mice were topically sensitized through application of CDDP before being challenged by oropharyngeal aspiration (OPA) with CDDP. Before and immediately after OPA challenge, pulmonary responses were assessed using whole body plethysmography (WBP). Results: CDDP did not induce an immediate response or alter the respiratory rate in sensitized mice. Two days later, baseline enhanced pause (Penh) values were significantly elevated (p < 0.05) in mice challenged with CDDP. When challenged with methacholine (Mch) aerosol, Penh values were significantly elevated (p < 0.05) in sensitized mice and respiratory rate was reduced (p < 0.05). Lymph node cell counts and immunoglobulin E levels also indicated successful sensitization to CDDP. Irrespective of the sensitization state of the mice, the number of neutrophils increased significantly in bronchoalveolar lavage fluid (BALF) following CDDP challenge. BALF from sensitized mice also contained 2.46 (±0.8) × 104 eosinophils compared to less than 0.48 (±0.2) × 104 cells in non-sensitized mice (p < 0.05). Conclusions: The results from this study indicate that dermal exposure to CDDP induces immunological changes consistent with type I hypersensitivity and that a single respiratory challenge is enough to trigger pulmonary responses in dermally sensitized mice. These data provide previously unknown insights into the mechanisms of CDDP hypersensitivity.

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

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

Small airway inflammation is associated with residual airway hyperresponsiveness in Th2-high asthma.

Background: Asthma is characterized by airway inflammation, variable airflow obstruction, and airway hyperresponsiveness (AHR). Generally, AHR takes longer to resolve than does airflow obstruction or clinical symptoms. AHR occasionally persists despite adequate asthma treatment.Objective: To evaluate factors which associates with residual AHR in patients with seemingly remitted airway inflammation.Methods: Patients who exhibited high fractional exhaled nitric oxide (FeNO) levels (>25 ppb) at the first visit (Visit 1) and normalized FeNO levels (<25 ppb) after adequate asthma treatment, including inhaled corticosteroid administration (Visit 2), were analyzed. Patients underwent a blood test, FeNO and small airway/alveolar nitric oxide concentration (CANO) measurements and a methacholine challenge test (continuous inhalation method) at both visits. Clinical indices were compared between patients with and without residual AHR.Results: Fifty patients were analyzed. All exhibited high FeNO levels at Visit 1 [mean, 54.0 ppb (95% confidence interval, 42.4-65.5)] and improvement of FeNO levels at Visit 2 [20.4 (19.2-21.6)] (p < 0.0001). Thirty-three patients (66%) had remission of AHR at Visit 2. No significant differences were observed between patients with and without residual AHR in terms of FeNO levels, lung function parameters and blood eosinophil counts at both visits. CANO level at Visit 2 was the only factor that significantly differed between patients with residual AHR [2.7 (1.9-3.6)] and those who achieved AHR remission [0.8 (0.5-1.0)] (p < 0.0001).Conclusion: Small airway inflammation, as assessed by CANO, was associated with residual AHR in patients with Th2-high asthma.

Modulation of protective reflex cough by acute immune driven inflammation of lower airways in anesthetized rabbits.

Chronic irritating cough in patients with allergic disorders may reflect behavioral or reflex response that is inappropriately matched to the stimulus present in the respiratory tract. Such dysregulated response is likely caused by sensory nerve damage driven by allergic mediators leading to cough hypersensitivity. Some indirect findings suggest that even acid-sensitive, capsaicin-insensitive A-δ fibers called "cough receptors" that are likely responsible for protective reflex cough may be modulated through immune driven inflammation. The aim of this study was to find out whether protective reflex cough is altered during acute allergic airway inflammation in rabbits sensitized to ovalbumin. In order to evaluate the effect of such inflammation exclusively on protective reflex cough, C-fiber mediated cough was silenced using general anesthesia. Cough provocation using citric acid inhalation and mechanical stimulation of trachea was realized in 16 ovalbumin (OVA) sensitized, anesthetized and tracheotomised rabbits 24h after OVA (OVA group, n = 9) or saline challenge (control group, n = 7). Number of coughs provoked by citric acid inhalation did not differ between OVA and control group (12,2 ±6,1 vs. 17,9 ± 6,9; p = 0.5). Allergic airway inflammation induced significant modulation of cough threshold (CT) to mechanical stimulus. Mechanically induced cough reflex in OVA group was either up-regulated (subgroup named "responders" CT: 50 msec (50-50); n = 5 p = 0.003) or down-regulated (subgroup named "non responders", CT: 1200 msec (1200-1200); n = 4 p = 0.001) when compared to control group (CT: 150 msec (75-525)). These results advocate that allergen may induce longer lasting changes of reflex cough pathway, leading to its up- or down-regulation. These findings may be of interest as they suggest that effective therapies for chronic cough in allergic patients should target sensitized component of both, reflex and behavioral cough.

Role of chemical composition and redox modification of poorly soluble nanomaterials on their ability to enhance allergic airway sensitisation in mice.

BACKGROUND: Engineered nanoparticles (NPs) have been shown to enhance allergic airways disease in mice. However, the influence of the different physicochemical properties of these particles on their adjuvant properties is largely unknown. Here we investigate the effects of chemical composition and redox activity of poorly soluble NPs on their adjuvant potency in a mouse model of airway hypersensitivity. RESULTS: NPs of roughly similar sizes with different chemical composition and redox activity, including CeO2, Zr-doped CeO2, Co3O4, Fe-doped Co3O4(using Fe2O3 or Fe3O4) and TiO2 NPs, all showed adjuvant activity. OVA induced immune responses following intranasal exposure of BALB/c mice to 0.02% OVA in combination with 200 µg NPs during sensitization (on day 1, 3, 6 and 8) and 0.5% OVA only during challenge (day 22, 23 and 24) were more pronounced compared to the same OVA treatment regime without NPs. Changes in OVA-specific IgE and IgG1 plasma levels, differential cell count and cytokines in bronchoalveolar lavage fluid (BALF), and histopathological detection of mucosa cell metaplasia and eosinophil density in the conducting airways were observed. Adjuvant activity of the CeO2 NPs was primarily mediated via the Th2 response, while that of the Co3O4 NPs was characterised by no or less marked increases in IgE plasma levels, BALF IL-4 and IL-5 concentrations and percentages of eosinophils in BALF and more pronounced increases in BALF IL-6 concentrations and percentages of lymphocytes in BALF. Co-exposure to Co3O4 NPs with OVA and subsequent OVA challenge also induced perivascular and peribronchiolar lymphoid cell accumulation and formation of ectopic lymphoid tissue in lungs. Responses to OVA combined with various NPs were not affected by the amount of doping or redox activity of the NPs. CONCLUSIONS: The findings indicate that chemical composition of NPs influences both the relative potency of NPs to exacerbate allergic airway sensitization and the type of immune response. However, no relation between the acellular redox activity and the observed adjuvant activity of the different NPs was found. Further research is needed to pinpoint the precise physiological properties of NPs and biological mechanisms determining adjuvant activity in order to facilitate a safe-by-design approach to NP development.

Association of Rhinovirus C Bronchiolitis and Immunoglobulin E Sensitization During Infancy With Development of Recurrent Wheeze.

Importance: Rhinovirus infection in early life, particularly with allergic sensitization, is associated with higher risks of developing recurrent wheeze and asthma. While emerging evidence links different rhinovirus species (eg, rhinovirus C) to a higher severity of infection and asthma exacerbation, to our knowledge, little is known about longitudinal associations of rhinovirus C infection during infancy with subsequent morbidities. Objective: To examine the association of different viruses (respiratory syncytial virus [RSV], rhinovirus species) in bronchiolitis with risks of developing recurrent wheeze. Design, Setting, and Participants: This multicenter prospective cohort study of infants younger than 1 year who were hospitalized for bronchiolitis was conducted at 17 hospitals across 14 US states during 3 consecutive fall to winter seasons (2011-2014). Exposures: Major causative viruses of bronchiolitis, including RSV (reference group) and 3 rhinovirus species (rhinovirus A, B, and C). Main Outcomes and Measures: Development of recurrent wheeze (as defined in national asthma guidelines) by age 3 years. Results: This analytic cohort comprised 716 infants who were hospitalized for RSV-only or rhinovirus bronchiolitis. The median age was 2.9 months (interquartile range, 1.6-3.8 months), 541 (76%) had bronchiolitis with RSV only, 85 (12%) had rhinovirus A, 12 (2%) had rhinovirus B, and 78 (11%) had rhinovirus C infection. Overall, 231 (32%) developed recurrent wheeze by age 3 years. In the multivariable Cox model, compared with infants with RSV-only infection, the risk of recurrent wheeze was not significantly different in those with rhinovirus A or B (rhinovirus A: hazard ratio [HR], 1.27; 95% CI, 0.86-1.88; rhinovirus B: HR, 1.39; 95% CI, 0.51-3.77; both P > .10). By contrast, infants with rhinovirus C had a significantly higher risk (HR, 1.58; 95% CI, 1.08-2.32). There was a significant interaction between virus groups and IgE sensitization on the risk of recurrent wheeze (P for interaction < .01). Only infants with both rhinovirus C infection and IgE sensitization (to food or aeroallergens) during infancy had significantly higher risks of recurrent wheeze (HR, 3.03; 95% CI, 1.20-7.61). Furthermore, compared with RSV-only, rhinovirus C infection with IgE sensitization was associated with significantly higher risks of recurrent wheeze with subsequent development of asthma at age 4 years (HR, 4.06; 95% CI, 1.17-14.1). Conclusions and Relevance: This multicenter cohort study of infants hospitalized for bronchiolitis demonstrated between-virus differences in the risk of developing recurrent wheeze. Infants with rhinovirus C infection, along with IgE sensitization, had the highest risk. This finding was driven by the association with a subtype of recurrent wheeze: children with subsequent development of asthma.

Epicutaneous Exposure to Peanut Oil Induces Systemic and Pulmonary Allergic Reaction in Mice.

BACKGROUND: The prevalence of peanut allergy (PA) is constantly on the rise. Atopic dermatitis (AD) is a major risk factor for developing food allergy. Some bath oils and skin creams used for treating AD contain peanut oil, and it has been suggested that exposure to peanut allergens through a disrupted skin barrier is a potential cause of PA. Our aim was to investigate whether application of peanut oil to irritated skin causes a systemic or respiratory allergic response to peanuts in an animal model. METHODS: BALB/c mice underwent epicutaneous sensitization with either peanut oil (PM, n = 9) or phosphate buffered solution (controls, n = 9) daily for 5 consecutive days. Ten days after the last exposure the mice were challenged with intranasal peanut protein for 5 consecutive days. Bronchial alveolar lavage fluid was collected for cellular studies and measurement of cytokine levels. Sera were collected for immunoglobulin E (IgE) measurement. RESULTS: Epicutaneous peanut oil sensitization increased leukocyte and eosinophil counts and interleukin-13 levels (p = 0.003, p = 0.0006 and p = 0.03, respectively), in addition to increasing total serum IgE (p = 0.03). CONCLUSIONS: The results suggest that topical application of peanut oil may play a role in the etiology of PA.

House Dust Mite-Induced Allergic Lung Inflammation Is Not Exacerbated in Sickle Cell Disease Mice.

AIM: Asthma appears to be a common comorbid condition in children with sickle cell disease (SCD), and such individuals may be at a higher risk for increased morbidity and mortality. However, several reports have indicated that asthma severity is not particularly high in those with SCD, and airway hyperreactivity and wheeze may be independently associated with SCD. In SCD mice, exacerbated allergic airway disease (AAD) has been observed in response to the model antigen ovalbumin (OVA). We sought to determine if allergic lung inflammation is also exacerbated in SCD mice when they are exposed to the human allergen, house dust mite (HDM). METHODS AND RESULTS: Eosinophil counts in bronchoalveolar lavage fluid were determined by cytocentrifugation and increased in both wild-type (WT) and SCD mice after acute exposure to a high dose (25 µg) of HDM, which then decreased in chronically exposed mice. WT mice exposed to a low dose of HDM (1 µg) followed the same pattern of eosinophil flux, but SCD mice did not induce much eosinophilia after acute exposure to HDM. As was observed in previous studies, lung lesions similarly increased in severity in both WT and SCD mice after acute exposure to HDM, which remained elevated after chronic exposure. Furthermore, serum HDM-specific IgE titers similarly increased and selected serum cytokines were similar in both WT and SCD mice. CONCLUSION: These results contrast with previous reports of exacerbated AAD in SCD mice exposed to OVA and support the alternative hypothesis that asthmatic responses are normal in those with SCD.

Experimental protocol for development of adjuvant-free murine chronic model of allergic asthma.

BACKGROUND: Mouse models of allergic asthma play a crucial role in exploring of asthma pathogenesis and testing of novel anti-inflammatory drugs. Widely used acute asthma models usually developed with adjuvant (aluminum hydroxide (alum)) do not reproduce one of the main asthma feature - airway remodeling while chronic asthma model mimic the pathophysiology of human disease. Moreover, the use of alum causes distress in experimental animals and impedes the test of adjuvant-containing drugs. In this study, we aimed to develop a chronic adjuvant-free asthma model with pronounced asthmatic phenotype. METHODS: Female BALB/c mice were divided into 3 groups. The first group was sensitized with intraperitoneal injections of ovalbumin (OVA) emulsified in aluminum hydroxide on days 0, 14, 28 followed by two stages of intranasally challenge with OVA on days 41-43 and 62-64. The second group was subcutaneously sensitized with the same dose of OVA without adjuvant and challenged on the same days. The third group (negative control) included mice which did not received any kind of treatment (i.e. sensitization and challenge). Serum levels of OVA-specific IgE, IgG2a and IgG1 antibodies were detected by ELISA. Airway hyper-responsiveness was measured by non-invasive plethysmography on days 44 and 65. Bronchoalveolar lavage fluids (BALF) sampled in all groups on days 45 and 66 were analyzed by light microscopy. The left lung was removed for histological analysis. The IL-4 and IFNγ mRNA expression in BALF cells was evaluated by RT-PCR. RESULTS: The OVA-specific IgE antibody response was two-fold increased in mice from adjuvant-free group compared to the adjuvant group that reflects reorientation of immune response towards Th2 phenotype. At the same time, the level of OVA-specific IgG1 and IgG2a antibodies was increased in the adjuvant group. Airway hyperresponsiveness to methacholine in mice of both experimental groups was two-fold higher than in control. Analysis of cell composition in BAL has shown a significant increase in eosinophil count in both experimental groups that indicate the development of allergic inflammation. Lung histology revealed airway remodeling in both experimental groups including goblet cell hyperplasia/metaplasia, thickening of airway walls, collagen deposition in the wall of distal airways. Additionally, the tendency to develop hypertrophy of bronchial smooth muscle layer was observed. Study of gene expression in BAL cells revealed the increase of IL-4 level in both adjuvant and adjuvant-free groups while IFNγ expression in both experimental groups was similar to control group. CONCLUSION: We have developed a chronic adjuvant-free mouse asthma model which possesses all necessary features of the disease including airway remodeling and is more suitable for pre-clinical evaluation of novel therapeutic approaches including adjuvant-containing drugs.

Is There an Overlap in Immune Response Between Allergic Bronchopulmonary and Chronic Pulmonary Aspergillosis?

BACKGROUND: Chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA) are presumed to represent 2 distinct manifestations of Aspergillus species in the lung. OBJECTIVE: To investigate any possible overlap of the immunological tests used for diagnosing ABPA in proven cases of CPA. METHODS: In consecutive subjects with CPA, we calculated the proportion of subjects who tested positive for all the immunological investigations used to diagnose ABPA (Aspergillus fumigatus specific IgE >0.35 kUA/L, total IgE ≥500 IU/mL, and eosinophil count ≥500 cells/µL) or obligatory criteria (A. fumigatus specific IgE >0.35 kUA/L and total IgE ≥500 IU/mL). RESULTS: A total of 269 subjects (53.5% males) of CPA with the mean (standard deviation [SD]) age of 44.3 (14.7) years were enrolled. The most common underlying disease was previously treated pulmonary tuberculosis (n = 230, 85.5%). Ninety-three (34.6%) subjects had total IgE ≥500 IU/mL, whereas A. fumigatus specific IgE >0.35 kUA/L was seen in 112 (41.6%) subjects. Thirteen (4.8%) subjects met all the immunological criteria for ABPA, whereas 59 (21.9%) subjects met the obligatory criteria. Subjects meeting the obligatory criteria had significantly higher eosinophil count (P ≤ .0001), greater immediate cutaneous reactivity to Aspergillus antigen (CPA-others vs obligatory criteria, 9.8 ± 13.9 vs 13.9 ± 14.9 mm, P value = .048), higher A. fumigatus specific IgG (99.3 ± 61.9 vs 122 ± 66.6 mgA/L, P = .015), and greater number of fungal balls (0.9 ± 0.7 [range, 0-3] vs 1.1 ± 0.9 [range, 0-4], P = .026) compared with those without. CONCLUSIONS: Approximately 5% of subjects with CPA fulfilled all the immunological criteria used for diagnosing ABPA, whereas 22% met the obligatory criteria for ABPA. Whether these patients would require a different management protocol requires further investigation.

Identification of airborne pollen allergens from two avenue trees of India.

An attempt has been made to detect airborne pollen of Lagerstroemia speciosa (LS) and Spathodea campanulata (SC) - two common avenue trees of India as potential sources of aeroallergens and also to identify the major IgE-reactive components present in them. The airborne pollen concentration was assessed using a Burkard sampler. A detailed questionnaire on clinical data of 1490 patients was recorded based on hospital data. We assessed the allergenicity of pollen by in vivo and in vitro tests. The correlation among meteorological factors, pollen seasons and allergenic potency of patients was assessed by multiple regression analysis. The sensitivity of patients to pollen antigens was highly correlated with pollen seasons. In SDS-PAGE, 15 protein bands were detected from LS pollen, while 14 bands from SC. The IgE-specific immunoblotting with patients' sera allergic to LS displayed five major allergens, while four major allergens were detected from SC. This would be the first report from India to prove the allergenic potentiality of airborne pollen of these two common avenue trees of India.

Evaluation of Sensitivity Toward Storage Mites and House Dust Mites Among Nasobronchial Allergic Patients of Kolkata, India.

House dust mites (HDMs) are the major constituents of house dust (HD). HD and HDM sensitization is well documented worldwide. Storage mite (SM) sensitization is presently lacking from India. The present study evaluated the sensitization of both HDM and SM among 372 allergic rhinitis patients reported to the Allergy and Asthma Research Center of Kolkata metropolitan, India. HD samples were collected from the patients' home and analyzed for the major constituent mites. HD and six constituent mites Dermatophagoides pteronyssinus (DP), Dermatophagoides farina Hughes (Acari: Pyroglyphidae) (DF), Blomia tropicalis (BT), Acarus siro Linnaeus (Acari: Acaridae) (AS), Lepidoglyphus destructor (LD), and Tyrophagus putrescentiae (Schrank) (Acari: Acaridae) (TP) are tested for the allergenic potential through Skin Prick Test (SPT). Three SMs, namely AS, LD, and TP, were newly included in the mite SPT extract for the first time in Kolkata. In total, 330 patients showed significant positive SPT toward any one allergen tested. HD was the major elicitor exhibiting 92.42% response. Individuals of age group 15-40 were the worst sufferers. DF showed the highest sensitization (87.87%) among the dust mites. The SMs also contributed significantly to prove their sensitizing potential. SPT rates for AS, LD, and TP were 33, 25, and 18%, respectively. SPT grades and total Immunoglobulin E (IgE) were positively correlated for each of the allergens. Most of the patients were multi-sensitized (95%) and represented markedly high total IgE levels (>500 IU/ml). Three SMs proved to be significant allergens for the studied population. The sensitization toward these SMs is first time reported from India and can be recommended for inclusion of routine SPT for better outcome in the future.

Maternal Separation as Early-Life Stress Causes Enhanced Allergic Airway Responses by Inhibiting Respiratory Tolerance in Mice.

Epidemiologic studies indicate that exposure to psychosocial stress in early childhood is a risk factor of adult-onset asthma, but the mechanisms of this relationship are poorly understood. Therefore, we examined whether early-life stress increases susceptibility to adult-onset asthma by inhibiting the development of respiratory tolerance. Neonatal BALB/c female mice were aerosolized with ovalbumin (OVA) to induce immune tolerance prior to immune sensitization with an intraperitoneal injection of OVA and the adjuvant aluminum hydroxide. Maternal separation (MS) was applied as an early-life stressor during the induction phase of immune tolerance. The mice were challenged with OVA aerosol in adulthood, and allergic airway responses were evaluated, including airway hyper-responsiveness to inhaled methacholine, inflammatory cell infiltration, bronchoalveolar lavage fluid levels of interleukin (IL)-4, IL-5, and IL-13, and serum OVA-specific IgE. We then evaluated the effects of MS on the development of regulatory T (Treg) cells in bronchial lymph nodes (BLN) and on splenocyte proliferation and cytokine expression. In mice that underwent MS and OVA tolerization, the allergic airway responses and OVA-induced proliferation and IL-4 expression of splenocytes were significantly enhanced. Furthermore, exposure to MS was associated with a lower number of Treg cells in the BLN. These findings suggest that exposure to early-life stress prevents the acquisition of respiratory tolerance to inhaled antigen due to insufficient Treg cell development, resulting in Th2-biased sensitization and asthma onset. We provide the evidence for inhibitory effects of early-life stress on immune tolerance. The present findings may help to clarify the pathogenesis of adult-onset asthma.