An initial assessment of the involvement of transglutaminase2 in eosinophilic bronchitis using a disease model developed in C57BL/6 mice.

The detailed pathogenesis of eosinophilic bronchitis (EB) remains unclear. Transglutaminase 2 (TG2) has been implicated in many respiratory diseases including asthma. Herein, we aim to assess preliminarily the relationship of TG2 with EB in the context of the development of an appropriate EB model through ovalbumin (OVA) sensitization and challenge in the C57BL/6 mouse strain. Our data lead us to propose a 50 µg dose of OVA challenge as appropriate to establish an EB model in C57BL/6 mice, whereas a challenge with a 400 µg dose of OVA significantly induced asthma. Compared to controls, TG2 is up-regulated in the airway epithelium of EB mice and EB patients. When TG2 activity was inhibited by cystamine treatment, there were no effects on airway responsiveness; in contrast, the lung pathology score and eosinophil counts in bronchoalveolar lavage fluid were significantly increased whereas the cough frequency was significantly decreased. The expression levels of interleukin (IL)-4, IL-13, IL-6, mast cell protease7 and the transient receptor potential (TRP) ankyrin 1 (TRPA1), TRP vanilloid 1 (TRPV1) were significantly decreased. These data open the possibility of an involvement of TG2 in mediating the increased cough frequency in EB through the regulation of TRPA1 and TRPV1 expression. The establishment of an EB model in C57BL/6 mice opens the way for a genetic investigation of the involvement of TG2 and other molecules in this disease using KO mice, which are often generated in the C57BL/6 genetic background.

Development and immunogenic potentials of chitosan-saponin encapsulated DNA vaccine against avian infectious bronchitis coronavirus.

Infectious Bronchitis (IB) is an economically important avian disease that considerably threatens the global poultry industry. This is partly, as a result of its negative consequences on egg production, weight gain as well as mortality rate.The disease is caused by a constantly evolving avian infectious bronchitis virus whose isolates are classified into several serotypes and genotypes that demonstrate little or no cross protection. In order to curb the menace of the disease therefore, broad based vaccines are urgently needed. The aim of this study was to develop a recombinant DNA vaccine candidate for improved protection of avian infectious bronchitis in poultry. Using bioinformatics and molecular cloning procedures, sets of monovalent and bivalent DNA vaccine constructs were developed based on the S1 glycoprotein from classical and variants IBV strains namely, M41 and CR88 respectively. The candidate vaccine was then encapsulated with a chitosan and saponin formulated nanoparticle for enhanced immunogenicity and protective capacity. RT-PCR assay and IFAT were used to confirm the transcriptional and translational expression of the encoded proteins respectively, while ELISA and Flow-cytometry were used to evaluate the immunogenicity of the candidate vaccine following immunization of various SPF chicken groups (A-F). Furthermore, histopathological changes and virus shedding were determined by quantitative realtime PCR assay and lesion scoring procedure respectively following challenge of various subgroups with respective wild-type IBV viruses. Results obtained from this study showed that, groups vaccinated with a bivalent DNA vaccine construct (pBudCR88-S1/M41-S1) had a significant increase in anti-IBV antibodies, CD3+ and CD8+ T-cells responses as compared to non-vaccinated groups. Likewise, the bivalent vaccine candidate significantly decreased the oropharyngeal and cloacal virus shedding (p < 0.05) compared to non-vaccinated control. Chickens immunized with the bivalent vaccine also exhibited milder clinical signs as well as low tracheal and kidney lesion scores following virus challenge when compared to control groups. Collectively, the present study demonstrated that bivalent DNA vaccine co-expressing dual S1 glycoprotein induced strong immune responses capable of protecting chickens against infection with both M41 and CR88 IBV strains. Moreso, it was evident that encapsulation of the vaccine with chitosan-saponin nanoparticle further enhanced immune responses and abrogates the need for multiple booster administration of vaccine. Therefore, the bivalent DNA vaccine could serve as efficient and effective alternative strategy for the control of IB in poultry.

Prevalence of food allergen and aeroallergen sensitization among children in Sichuan province.

Food allergen and aeroallergen sensitization are common allergic diseases worldwide, with widely varying estimates of prevalence in children. Our study investigated the characteristics of ingestion and inhalation allergy among children from Sichuan province in Southwest China, so as to get public awareness of these disorders.A total of 1722 children between 0 and 14 years' old were enrolled in this study. They were outpatients in the West China Second University Hospital during June 2019 to September 2019. Serum specific IgE specific to 10 types of food allergen and 10 types of aeroallergen were estimated. Nutrition indicators were tested by electrochemical luminescence.59.70% children were allergic to at least 1 allergen, comprising 24.90% to aeroallergen and 38.81% to food allergen, respectively, whereas 36.28% children were allergic to both aeroallergen and food allergen. Milk was the most common food allergen, and egg came in second place. With regard to aeroallergen, house dust mite held the maximum proportion (65.02%), whereas dust mite followed behind. Inhalation allergy was more commonly seen in boys than girls. Bronchitis was the most common symptom of both allergies. In addition, the highest incidence age for children to be sensitive to food allergen and aeroallergen were 0∼2 years' old and 3∼5 years' old, respectively. It is worth mentioning that there was no significant difference in nutritional status between children with or without allergic diseases.Our findings reveal that milk, egg, house dust mite, and dust mite are the most common allergens among children in Sichuan province. Boys are more susceptible to aeroallergen than girls. Furthermore, the prevalence of ingestion and inhalation allergy varies from different age groups, and has no correlation with nutritional status. In brief, the analysis of the pattern of food allergen and aeroallergen sensitization is invaluable to effective diagnosis and treatment of allergic diseases.

Development and validation of a bronchoscopically defined bronchitis scoring tool in children.

INTRODUCTION/AIM: A validated tool for scoring bronchitis during flexible bronchoscopy (FB) is potentially useful for clinical practice and research. We aimed to develop a bronchoscopically defined bronchitis scoring system in children (BScore) based on our pilot study. METHODS: Children undergoing FB were prospectively enrolled. Their FB was digitally recorded and assessed (two clinicians blinded to each other and clinical history) for six features: secretion amount (six-point scale), secretion color (BronkoTest, 0-8), mucosal oedema (0-3), ridging (0-3), erythema (0-3), and pallor (0-3) based on pre-determined criteria. We correlated (Spearman's rho) each feature with bronchoalveolar lavage (BAL) neutrophil percentage (neutrophil%). BScore was then derived using models with combinations of the six features that best related to airway BAL neutrophil%. The various models of BScore were plotted against BAL neutrophil% using receiver operating characteristic (ROC) curves. RESULTS: We analyzed 142 out of 150 children enrolled. Eight children were excluded for unavailability of BAL cytology or FB recordings. Chronic/recurrent cough was the commonest indication for FB (75%). The median age was 3 years (IQR, 1.5-5.3 years). Secretion amount (r = 0.42) and color (r = 0.46), mucosal oedema (r = 0.42), and erythema (r = 0.30) significantly correlated with BAL neutrophil%, P < .0001. The highest area under ROC (aROC) was obtained by the addition of the scores of all features excluding pallor (aROC = 0.84; 95% CI, 0.76-0.90) with airway neutrophilia (defined as BAL neutrophil% of >10%). CONCLUSION: This prospective study has developed the first validated bronchitis scoring tool in children based on bronchoscopic visual inspection of airways. Further validation in other cohorts is however required.

A 27-year-old man with recurrent sinopulmonary and cutaneous infections.

The increasing availability of genetic testing for modern immunologists in the evaluation of immune diseases could provide a definite diagnosis in elusive cases. A 27-year-old white male patient presented to the clinic with recurrent sinopulmonary and cutaneous infections since childhood. The patient's mother had seronegative polyarthritis, and one of two sisters of the patient had chronic sinopulmonary infections. Serum immunoglobulins, immunoglobulin G (IgG) subclasses, lymphocyte subset markers, mannose-binding lectin, mitogen and antigen stimulation, bacteriophage study, and Streptococcus pneumoniae titers to 23 serotypes were all normal. B-cell phenotyping revealed a decrease in both nonswitched memory B cells (CD19+CD27+IgD+) and switched memory B-cells (CD19+CD27+IgD-). Genetic testing and the improvement of clinical symptoms after IgG replacement led to the final diagnosis.

Response to bronchodilator and clinical, pathophysiological features in patients with nonasthmatic eosinophilic bronchitis.

INTRODUCTION: Whether nonasthmatic eosinophilic bronchitis (NAEB) shows response to bronchodilator (RB) remains unclear. OBJECTIVES: To investigate the RB and its relationship with clinical and pathophysiological features in NAEB. METHODS: Fifty-one patients with NAEB were assigned in a 2:1 ratio to receive oral bambuterol hydrochloride (n = 34, 10 mg, once daily, for 3 days) or matched placebo (n = 17) randomly, of whom 48 patients (32 with bronchodilator and 16 with placebo) completed the study. Sputum induction, spirometry and cough reflex sensitivity were measured. RB was considered when cough Visual analogue scale (VAS) score decreased 30% or more after treatment. Cough reflex sensitivity was defined as the lowest concentration of capsaicin inducing five coughings or more (C5), and presented as Log C5. RESULTS: The responsive rate of patients with bronchodilator was significantly higher than that with placebo (34.4% vs 6.3%, P < 0.05). The VAS score decreased significantly in patients with bronchodilator (median: 6.0-3.0, P < 0.01). There was a significantly higher median Log C5 (2.7 vs 1.3, P < 0.05), and a higher trend of decline in FEV1 % predicted and MMEF% predicted after bronchial provocation in patients with RB as compared with patients without RB. No significant differences in baseline percentages of sputum eosinophil were found between patients with RB and that without RB. CONCLUSIONS: One third of patients with NAEB respond well to bronchodilator treatment, which are related with lower cough reflex sensitivity and increased airway responsiveness. The relationship between NAEB and asthma needs to be investigated further.

The Effect of Flavored E-cigarettes on Murine Allergic Airways Disease.

Flavored e-cigarettes are preferred by the majority of users yet their potential toxicity is unknown. Therefore our aim was to determine the effect of selected flavored e-cigarettes, with or without nicotine, on allergic airways disease in mice. Balb/c mice were challenged with PBS or house dust mite (HDM) (Days 0, 7, 14-18) and exposed to room air or e-cigarette aerosol for 30 min twice daily, 6 days/week from Days 0-18 (n = 8-12/group). Mice were exposed to Room Air, vehicle control (50%VG/%50PG), Black Licorice, Kola, Banana Pudding or Cinnacide without or with 12 mg/mL nicotine. Mice were assessed at 72 hours after the final HDM challenge. Compared to mice challenged with HDM and exposed to Room Air, nicotine-free Cinnacide reduced airway inflammation (p = 0.045) and increased peripheral airway hyperresponsiveness (p = 0.02), nicotine-free Banana Pudding increased soluble lung collagen (p = 0.049), with a trend towards increased airway inflammation with nicotine-free Black Licorice exposure (p = 0.089). In contrast, all e-cigarettes containing nicotine suppressed airway inflammation (p < 0.001 for all) but did not alter airway hyperresponsiveness or airway remodeling. Flavored e-cigarettes without nicotine had significant but heterogeneous effects on features of allergic airways disease. This suggests that some flavored e-cigarettes may alter asthma pathophysiology even when used without nicotine.

Analysis of the Clinical Features of Tracheobronchial Fungal Infections with Tumor-Like Lesions.

BACKGROUND: Tracheobronchial fungal infections (TBFI) cause life-threatening complications in immunocompromised hosts but are rarely reported. Misdiagnosis and delayed antifungal treatment are associated with the high mortality rate of patients with TBFI. OBJECTIVES: This study analyzed the bronchoscopic features of TBFI and their roles in the early diagnosis of TBFI. METHODS: The demographic, clinical, radiologic, and bronchoscopic data of 53 patients diagnosed with TBFI in our department during a 15-year period were retrospectively analyzed. RESULTS: Most of the TBFI patients were male, and mass was the most common radiologic abnormality. Obvious predilection in primary bronchus distributions was observed. 41.9% of the 43 Aspergillus tracheobronchitis (AT) patients, 70% of the 10 tracheobronchial mucormycosis (TM) patients, and 100% of the 3 endobronchial cryptococcosis patients had been misdiagnosed as having cancer on bronchoscopy because of the presence of tumor-like lesions. The most common features of AT were bronchial occlusion with a mass or mucosal necrosis, bronchial stenosis with mucosal hyperplasia, or uneven mucosa. The main descriptions of TM were bronchial stenosis or obstruction due to mucosal necrosis, uneven mucosa, or a mass. The endoscopic characteristics of endobronchial cryptococcosis included occlusion due to uneven mucosa or mass, or external compressive stricture. CONCLUSION: Immunocompromised patients and immunocompetent patients with underlying disease displaying tumor-like lesions on bronchoscopy should be differentially diagnosed with cancer. Bronchial biopsy is indispensable for the early diagnosis of TBFI.

The Infectious Bronchitis Coronavirus Envelope Protein Alters Golgi pH To Protect the Spike Protein and Promote the Release of Infectious Virus.

Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro The E protein from avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane compared to wild-type (WT) IBV-infected cells, and aberrant cleavage of IBV S on virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.IMPORTANCE Coronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including the use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or antiviral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite for potential vaccines as well as in identifying novel antiviral therapeutics.

Recent thymic emigrants, T regulatory cells, and BAFF level in children with X-linked agammaglobulinaemia in association with chronic respiratory disease.

BACKGROUND: X-linked agammaglobulinaemia (XLA) is a genetic disorder affecting B cell maturation, which is characterised by a low number of B cells, agammaglobulinaemia and increased susceptibility to a variety of bacterial infections. This study was performed to assess T cell subpopulations in a group of children with XLA in association with chronic respiratory disease (CRD). METHODS: Numbers of T cell subpopulations (CD3+, CD4+, CD8+, CD3+DR+, naïve, memory, recent thymic emigrants (RTE), regulatory T cells, follicular T helpers) were measured by eight-colour flow cytometry in 22 XLA patients and 50 controls. BAFF level was measured by ELISA. RESULTS: XLA patients with CRD had a significantly lower percentage of RTE numbers and Tregs, while significantly higher absolute counts of lymphocytes, CD3+, CD8+, CD3+DR+ and CD4+CD45RO+ T cells were detected as compared with healthy controls. In patients with XLA without CRD, the number of follicular T helper cells was altered significantly (percentage and absolute), as compared with healthy controls. Additionally, they had significantly higher counts (percentage and absolute) of CD4+CD45RA+ cells and lower percentage of CD4+CD45RO+ cells in comparison with healthy controls. CONCLUSIONS: Our study affords new information concerning CRD and T cell subsets that differentiate or are maintained in the absence of B cells in children with XLA. T cell's homeostasis depends on the presence of chronic respiratory disease that may be caused by the delay in diagnosis.

Advanced Role of Neutrophils in Common Respiratory Diseases.

Respiratory diseases, always being a threat towards the health of people all over the world, are most tightly associated with immune system. Neutrophils serve as an important component of immune defense barrier linking innate and adaptive immunity. They participate in the clearance of exogenous pathogens and endogenous cell debris and play an essential role in the pathogenesis of many respiratory diseases. However, the pathological mechanism of neutrophils remains complex and obscure. The traditional roles of neutrophils in severe asthma, chronic obstructive pulmonary diseases (COPD), pneumonia, lung cancer, pulmonary fibrosis, bronchitis, and bronchiolitis had already been reviewed. With the development of scientific research, the involvement of neutrophils in respiratory diseases is being brought to light with emerging data on neutrophil subsets, trafficking, and cell death mechanism (e.g., NETosis, apoptosis) in diseases. We reviewed all these recent studies here to provide you with the latest advances about the role of neutrophils in respiratory diseases.

Increased sputum levels of thymus and activation-regulated chemokine in children with asthma not eosinophilic bronchitis

BACKGROUND: Thymus and activation-regulated chemokine (TARC), a member of the CC chemokine family, plays a crucial role in Th2-specific inflammation. We aimed to determine the concentration of sputum TARC in children with asthma and eosinophilic bronchitis (EB) and its relation with eosinophilic inflammation, pulmonary function, and bronchial hyper-responsiveness. METHODS: In total, 90 children with asthma, 38 with EB, and 45 control subjects were enrolled. TARC levels were measured in sputum supernatants using an ELISA. We performed pulmonary function tests and measured exhaled fractional nitric oxide, eosinophil counts in blood, and sputum and serum levels of total IgE in all children. RESULTS: Sputum TARC levels were significantly higher in children with asthma than in either children with EB (p = 0.004) or the control subjects (p = 0.014). Among patients with asthma, sputum TARC concentration was higher in children with sputum eosinophilia than in those without sputum eosinophilia (p = 0.035). Sputum TARC levels positively correlated with eosinophil counts in sputum, serum total IgE levels, exhaled fractional nitric, and the bronchodilator response. Negative significant correlations were found between sputum TARC and FEV1/FVC (the ratio of forced expiratory volume in one second and forced expiratory vital capacity) or PC20 (the provocative concentration of methacholine causing a 20% decrease in the FEV1). CONCLUSION: Elevated TARC levels in sputum were detected in children with asthma but not in children with EB. Sputum TARC could be a supportive marker for discrimination of asthma from EB in children showing characteristics of eosinophilic airway inflammation

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[Development of an eosinophilic bronchitis mouse model and the effects of eosinophil activation on airway hyperresponsiveness in eosinophilic bronchitis mice].

Objective: To develop a mouse model of eosinophilic bronchitis, and explore the effects of eosinophil activation on airway hyperresponsiveness. Methods: A total of 60 female specific pathogen free BALB/c female mice were divided randomly into four groups: polymyxin B group, normal saline group, polymyxin B+ N-methionine leucine phenylalanine (fMLP) group, normal saline+ fMLP group. All the groups were given corresponding nasal drops for 21 days. The former two groups were given 12 µl 0.5% polymyxin B or normal saline once a day by transnasal administration respectively. Besides the above, the latter two groups were given 10 µl fMLP (0.05 mg/ml, dissolved in acetic acid) once by transnasal administration 3 hours after polymyxin B or normal saline administration on the 21st day. Within 24 hours after the last transnasal administration, cells in bronchoalveolar lavage fluid (BALF) were collected and analyzed by absolute different cell counts. Airway responsiveness (inspiratory and expiratory airway resistance and lung compliance) to acetyl choline (Ach) were measured 3 hours after the last transnasal administration in the former two groups and 24 hours after the last transnasal administration in the latter two groups. Eosinophil peroxidase (EPX), eosinophil cationic protein (ECP) and eosinophil chemotactic factor (ECF) levels in serum, BALF and lung tissue were tested by emzyme linked immunosorbent assay (ELISA). HE and Chromotrope 2R staining of lung tissue were used to observe the infiltration of inflammatory cells. The morphology of low-density eosinophils was observed under electron microscope. Results: The total cell counts of BALF in polymyxin B group were significantly higher than the normal saline group [29.50 (3.25)×10(4)/ml vs 15.25 (2.25)×10(4)/ml, P<0.001], especially eosinophil counts [11.76 (6.02)×10(4)/ml vs 0.12 (1.08)×10(4)/ml, P<0.001]. However, no significant differences of inspiratory and expiratory airway resistance and lung compliance existed in the two groups. Inspiratory and expiratory airway resistance in polymyxin B+ fMLP group were significantly higher, lung compliance significantly lower than those in the other three groups (all P<0.001), and the EPX, ECP, ECF levels in serum, BALF and lung tissue of the polymyxin B+ fMLP group were significantly higher than the other three groups (all P<0.001). There were more inflammatory cells, especially eosinophils in lung tissue of polymyxin B and polymyxin B+ fMLP group. Meanwhile, more activated eosinophils in polymyxin B+ fMLP group were observed by electron microscopy. Conclusion: A mouse model of eosinophilic bronchitis can be successfully developed by transnasal administration of polymyxin B, and the eosinophil activation plays an important role in the occurrence of airway hyperresponsiveness.

Eotaxin and IL-4 levels are increased in induced sputum and correlate with sputum eosinophils in patients with nonasthmatic eosinophilic bronchitis.

Nonasthmatic eosinophilic bronchitis (NAEB) is characterized by chronic cough and airway eosinophilic inflammation. Airway and systemic inflammation cytokine profile have not been comprehensively described in patients with NAEB.The aim of the study was to identify the cytokine profile in sputum and serum of NAEB patients. Furthermore, the relationship between cytokines and clinical features would be evaluated.Induced sputum and serum were collected from untreated NAEB patients and healthy subjects. The cytokine profile in sputum and serum was analyzed by a bead-based multiplex cytokine assay including 21 cytokines.The levels of EGF, eotaxin, GM-CSF, GRO, IFN-γ, IL-1ß, IL-4, IL-6, IL-17A, IP-10, MIP-1α, and TNF-α in sputum were significantly higher in NAEB patients than that in healthy subjects (all P < 0.05). Values of area under the receiver operating characteristic curve (AUROC) of these cytokines were all above 0.750. The concentrations of eotaxin and IL-4 were positively correlated with sputum eosinophil percentage (r = 0.726, P = 0.002; r = 0.511, P = 0.043; respectively). No significant correlations between other cytokines (EGF, GM-CSF, GRO, IFN-γ, IL-1ß, IL-6, IL-17A, IP-10, MIP-1α, and TNF-α) in sputum and sputum eosinophil percentage were found. The level of IL-4 in serum was slightly higher in NAEB patients than in healthy subjects. However, there was no correlation between serum IL-4 levels and sputum eosinophil percentage.We identified the cytokine profile in sputum and serum from NAEB patients. Sputum eotaxin and IL-4 could have the potential to become the biomarkers for NAEB and might be useful to assist in the diagnosis of NAEB.