Combination of androgen and estrogen improves asthma by mediating Runx3 expression.

Objective: Asthma is a chronic heterogeneous airway disease, and imbalanced T-helper type 1 (Th1) and Th2 cell-mediated inflammation contribute to its pathogenesis. Although it has been suggested that androgen and estrogen were involved in development of asthma, the underlying mechanisms remained largely unclear. Studies have demonstrated that Runx3 could promote naive CD4+ T cells to differentiate into Th1 cells. Hence, our study aimed to explore the potential regulatory mechanism of androgen and estrogen on asthma via modulating Runx3. Methods: First, clinical assessments and pulmonary function tests were conducted on 35 asthma patients and 24 healthy controls. The concentrations of androgen, estrogen, and androgen estrogen ratios were assessed in peripheral blood samples of asthma patients and healthy controls. Then, a murine asthma model was established to explore the effects of estrogen and androgen (alone or in combination) on asthma. Third, an in vitro assay was used to explore the mechanism of combination of androgen and estrogen in asthma. Results: We observed decreased androgen and increased estrogen levels in asthma patients compared with healthy controls. In mice with experimental asthma, there were increased serum concentrations of estrogen and decreased serum concentrations of androgen, intervention with combination of androgen and estrogen alleviated airway inflammations, increased Runx3 expressions and elevated Th1 differentiation. In CD4+ T cells co-cultured with bronchial epithelial cells (BECs), treatment with androgen plus estrogen combination promoted Th1 differentiation, which was mitigated by Runx3 knockdown in BECs and enhanced by Runx3 overexpression. Conclusion: These findings suggest that androgen estrogen combination modulate the Th1/Th2 balance via regulating the expression of Runx3 in BECs, thereby providing experimental evidence supporting androgen and estrogen combination as a novel therapy for asthma.

Ratio of plasma IL-13/TNF- ∝ and CXCL10/CCL17 predicts mepolizumab and omalizumab response in asthma better than eosinophil count or immunoglobulin E level.

To date, most studies to identify biomarkers associated with response to the anti-interleukin 5 agent, mepolizumab, and to the anti-immunoglobulin E agent, omalizumab have focused on clinically available biomarkers, such as the peripheral blood eosinophil counts (BEC) and total immunoglobulin E (IgE). However, these biomarkers often have low predictive accuracy, with many patients with eosinophilic or allergic asthma failing to demonstrate clinical response to mepolizumab or omalizumab respectively. In this study, we evaluated the association of baseline pre-biologic plasma levels of 26 cytokines and chemokines, including T-helper 1 (Th1)-, Th2-, Th17-related cytokines, and their ratios with subsequent clinical response to mepolizumab or omalizumab. We defined clinical response as a reduction in the baseline annual exacerbation rate by half or more over the one-year period following initiation of the biologic. Baseline levels of plasma IL-13 were differentially elevated in responders versus non-responders to mepolizumab and plasma CXCL10 levels were differentially elevated in responders to omalizumab. The ratio of IL-13/TNF-α had the best sensitivity and specificity in predicting response to mepolizumab and CXCL10/CCL17 to omalizumab, and these performed better as predictive biomarkers of response than BEC and IgE. Cytokines and chemokines associated with airway eosinophilia, allergic inflammation, or Th2 inflammation, such as IL-13 and CXCL10, may be better predictors of clinical response to mepolizumab and omalizumab, than IL-5 or IgE, the targets of mepolizumab and omalizumab.

Galectin-10 in serum extracellular vesicles reflects asthma pathophysiology.

BACKGROUND: Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes. OBJECTIVE: We sought to identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs). METHODS: We performed data-independent acquisition of serum EVs from 4 healthy controls, 4 noneosinophilic asthma (NEA) patients, and 4 eosinophilic asthma (EA) patients to identify novel BMs for BA. We confirmed EA-specific BMs via data-independent acquisition validation in 61 BA patients and 23 controls. To further validate these findings, we performed data-independent acquisition for 6 patients with chronic rhinosinusitis without nasal polyps and 7 patients with chronic rhinosinusitis with nasal polyps. RESULTS: We identified 3032 proteins, 23 of which exhibited differential expression in EA. Ingenuity pathway analysis revealed that protein signatures from each phenotype reflected disease characteristics. Validation revealed 5 EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase, major basic protein, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. The potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnostic capability and detection of airway obstruction. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified in EVs and tissues from patients with chronic rhinosinusitis with nasal polyps, 5 (including Gal10 and eosinophil peroxidase) showed significant correlations between EVs and tissues. Gal10 release from EVs was implicated in eosinophil extracellular trapped cell death in vitro and in vivo. CONCLUSION: Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target for liquid biopsy approaches.

Persistent Asthma Is Associated With Carotid Plaque in MESA.

Background Asthma and atherosclerotic cardiovascular disease share an underlying inflammatory pathophysiology. We hypothesized that persistent asthma is associated with carotid plaque burden, a strong predictor of atherosclerotic cardiovascular disease events. Methods and Results The MESA (Multi-Ethnic Study of Atherosclerosis) enrolled adults free of known atherosclerotic cardiovascular disease at baseline. Subtype of asthma was determined at examination 1. Persistent asthma was defined as asthma requiring use of controller medications, and intermittent asthma was defined as asthma without controller medications. B-mode carotid ultrasound was performed to detect carotid plaques (total plaque score [TPS], range 0-12). Multivariable regression modeling with robust variances evaluated the association of asthma subtype and carotid plaque burden. The 5029 participants were a mean (SD) age of 61.6 (10.0) years (53% were women, 26% were Black individuals, 23% were Hispanic individuals, and 12% were Chinese individuals). Carotid plaque was present in 50.5% of participants without asthma (TPS, 1.29 [1.80]), 49.5% of participants with intermittent asthma (TPS, 1.25 [1.76]), and 67% of participants with persistent asthma (TPS, 2.08 [2.35]) (P≤0.003). Participants with persistent asthma had higher interleukin-6 (1.89 [1.61] pg/mL) than participants without asthma (1.52 [1.21] pg/mL; P=0.02). In fully adjusted models, persistent asthma was associated with carotid plaque presence (odds ratio, 1.83 [95% confidence interval, 1.21-2.76]; P<0.001) and TPS (ß=0.66; P<0.01), without attenuation after adjustment for baseline interleukin-6 (P=0.02) or CRP (C-reactive protein) (P=0.01). Conclusions Participants with persistent asthma had higher carotid plaque burden and higher levels of inflammatory biomarkers, compared with participants without asthma. Adjustment for baseline inflammatory biomarkers did not attenuate the association between carotid plaque and asthma subtype, highlighting the increased atherosclerotic cardiovascular disease risk among those with persistent asthma may be multifactorial.

The Association of Passive Smoking and Serum Urotensin-II Levels in Children.

Urotensin-II (UT-II) is the most powerful vasoconstrictor agent and is known to play a role in heart failure, diabetes, pulmonary hypertension and asthma. The effect of passive smoking on UT-II levels is unknown. The present study aims to evaluate serum UT-II levels in children exposed to passive smoke. The study included a total of 120 children; 47 children not exposed to passive smoke were included in Group 1 (control group), and 73 children exposed to passive smoke were included in Group 2. Serum samples of the participants were stored at -80 °C after centrifugation and were assessed at least two times with high-precision human ELISA kits. Serum UT-II levels were significantly higher in the children exposed to passive smoke than in the children not exposed. Furthermore, Group 2 was grouped according to the number of cigarettes smoked at home per day, type of passive smoking (second-hand smoke or third-hand smoke), and how many people in their family and/or living together smoked. There was a positive correlation between the number of cigarettes they were exposed to per day and serum UT-II levels. Passive smoking in childhood may be associated with high serum UT-II levels.

Serum Proteomic Analysis Identifies SAA1, FGA, SAP, and CETP as New Biomarkers for Eosinophilic Granulomatosis With Polyangiitis.

Background: Eosinophilic granulomatosis with polyangiitis (EGPA) is characterized by asthma-like attacks in its early stage, which is easily misdiagnosed as severe asthma. Therefore, new biomarkers for the early diagnosis of EGPA are needed, especially for differentiating the diagnosis of asthma. Objectives: To identify serum biomarkers that can be used for early diagnosis of EGPA and to distinguish EGPA from severe asthma. Method: Data-independent acquisition (DIA) analysis was performed to identify 45 healthy controls (HC), severe asthma (S-A), and EGPA patients in a cohort to screen biomarkers for early diagnosis of EGPA and to differentiate asthma diagnosis. Subsequently, parallel reaction monitoring (PRM) analysis was applied to a validation cohort of 71 HC, S-A, and EGPA patients. Result: Four candidate biomarkers were identified from DIA and PRM analysis-i.e., serum amyloid A1 (SAA1), fibrinogen-α (FGA), and serum amyloid P component (SAP)-and were upregulated in the EGPA group, while cholesteryl ester transfer protein (CETP) was downregulated in the EGPA group compared with the S-A group. Receiver operating characteristics analysis shows that, as biomarkers for early diagnosis of EGPA, the combination of SAA1, FGA, and SAP has an area under the curve (AUC) of 0.947, a sensitivity of 82.35%, and a specificity of 100%. The combination of SAA1, FGA, SAP, and CETP as biomarkers for differential diagnosis of asthma had an AUC of 0.921, a sensitivity of 78.13%, and a specificity of 100%, which were all larger than single markers. Moreover, SAA1, FGA, and SAP were positively and CETP was negatively correlated with eosinophil count. Conclusion: DIA-PRM combined analysis screened and validated four previously unexplored but potentially useful biomarkers for early diagnosis of EGPA and differential diagnosis of asthma.

Plasma proteome changes linked to late phase response after inhaled allergen challenge in asthmatics.

BACKGROUND: A subset of individuals with allergic asthma develops a late phase response (LPR) to inhaled allergens, which is characterized by a prolonged airway obstruction, airway inflammation and airway hyperresponsiveness. The aim of this study was to identify changes in the plasma proteome and circulating hematopoietic progenitor cells associated with the LPR following inhaled allergen challenge. METHODS: Serial plasma samples from asthmatics undergoing inhaled allergen challenge were analyzed by mass spectrometry and immunosorbent assays. Peripheral blood mononuclear cells were analyzed by flow cytometry. Mass spectrometry data were analyzed using a linear regression to model the relationship between airway obstruction during the LPR and plasma proteome changes. Data from immunosorbent assays were analyzed using linear mixed models. RESULTS: Out of 396 proteins quantified in plasma, 150 showed a statistically significant change 23 h post allergen challenge. Among the most upregulated proteins were three protease inhibitors: alpha-1-antitrypsin, alpha-1-antichymotrypsin and plasma serine protease inhibitor. Altered levels of 13 proteins were associated with the LPR, including increased factor XIII A and decreased von Willebrand factor. No relationship was found between the LPR and changes in the proportions of classical, intermediate, and non-classical monocytes. CONCLUSIONS: Allergic reactions to inhaled allergens in asthmatic subjects were associated with changes in a large proportion of the measured plasma proteome, whereof protease inhibitors showed the largest changes, likely to influence the inflammatory response. Many of the proteins altered in relation to the LPR are associated with coagulation, highlighting potential mechanistic targets for future treatments of type-2 asthma.

Soluble ACE2 and TMPRSS2 Levels in the Serum of Asthmatic Patients.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2) are key proteins mediating viral entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although gene expressions of ACE2 and TMPRSS2 have been analyzed in various organs and diseases, their soluble forms have been less studied, particularly in asthma. Therefore, we aimed to measure circulating ACE2 and TMPRSS2 in the serum of asthmatics and examine their relationship with clinical characteristics. METHODS: Clinical data and serum samples of 400 participants were obtained from an asthma cohort. The soluble ACE2 (sACE2) and soluble TMPRSS2 (sTMPRSS2) level was measured by enzyme-linked immunosorbent assay, and the values underwent a natural log transformation. Associations between sACE2 and TMPRSS2 levels and various clinical variables were analyzed. RESULTS: The patients younger than 70 years old, those with eosinophilic asthma (eosinophils ≥ 200 cells/µL), and inhaled corticosteroids (ICS) non-users were associated with higher levels of sACE2. Blood eosinophils and fractionated exhaled nitric oxide levels were positively correlated with serum ACE2. In contrast, lower levels of sTMPRSS2 were noted in patients below 70 years and those with eosinophilic asthma, while no association was noted between ICS use and sTMPRSS2. The level of sTMPRSS2 also differed according to sex, smoking history, coexisting hypertension, and forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio. The proportion of sputum neutrophils was positively correlated with sTMPRSS2, while the FEV1/FVC ratio reported a negative correlation with sTMPRSS2. CONCLUSION: The levels of ACE2 and TMPRSS2 were differently expressed according to age, ICS use, and several inflammatory markers. These findings suggest variable susceptibility and prognosis of SARS-CoV-2 infection among asthmatic patients.

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

Effect of vitamin D supplementation on total and allergen-specific IgE in children with asthma and low vitamin D levels.

BACKGROUND: Observational studies have yielded inconsistent findings for the relation between vitamin D level and total IgE or allergic sensitization. OBJECTIVE: To determine whether vitamin D supplementation reduces levels of total IgE and IgE to each of 2 common indoor allergens in children with asthma and low vitamin D levels. METHODS: Total IgE, IgE to Dermatophagoides pteronyssinus, and IgE to Blattella germanica were measured at the randomization and exit visits for 174 participants in the Vitamin D Kids Asthma Study, a multicenter, double-blind, randomized placebo-controlled trial of vitamin D3 supplementation (4000 IU/d) to prevent severe exacerbations in children with persistent asthma and vitamin D levels less than 30 ng/mL. Multivariable linear regression was used for the analysis of the effect of vitamin D supplementation on change in each IgE measure. RESULTS: Participants were followed for an average of 316 days. At the exit visit, more subjects in the vitamin D arm achieved a vitamin D level equal to or more than 30 ng/mL compared with those in the placebo arm (87% vs 30%; P < .001). In a multivariable analysis, vitamin D3 supplementation had no significant effect on change in total IgE, IgE to Dermatophagoides pteronyssinus, or IgE to Blattella germanica between the exit and randomization visits (eg, for log10 total IgE, ß = 0.007; 95% CI, -0.061 to 0.074; P = .85). CONCLUSIONS: Vitamin D supplementation, compared with placebo, has no significant effect on serum levels of total IgE, IgE to dust mite, or IgE to cockroach in children with asthma and low vitamin D levels.

Dysregulated Arginine Metabolism in Young Patients with Chronic Persistent Asthma and in Human Bronchial Epithelial Cells.

BACKGROUND: Recent metabolomics studies have found circulatory metabolism alterations in patients with asthma, indicating that altered metabolites played a significant role in asthma. However, the regulatory mechanisms in asthma, especially in young chronic persistent asthma remain underexplored. METHODS: In this study, a prospective cohort of 162 patients diagnosed of asthma admitted to the First Affiliated Hospital of Xi'an Jiaotong University from January 2018 to December 2019 was used to perform a nested case-control study. Among them, we included 30 patients with chronic persistent asthma between 20 to 35 years old; 30 health control with evenly distributed age and sex were then recruited. Nontargeted metabolomics was applied to identify serum metabolic profiles and altered metabolic pathways. RESULTS: In vitro, human bronchial epithelial cells (HBECs) line BEAS-2B with the addition of L-citrulline and/or asymmetric dimethylarginine (ADMA) model was utilized and the concentrations of nitric oxide (NO) metabolites were tested to evaluate the therapeutic potential of L-citrulline. The young patients with chronic persistent asthma displayed dysregulated serum metabolic profiles, especially enriched in arginine metabolism. The ratio of L-citrulline to ornithine is associated with blood eosinophil count. In vitro, adding L-citrulline could reverse ADMA-mediated reduction of NOx at lower L-arginine concentration (25 µM), but was ineffective in the higher L-arginine concentration (100 µM) media. CONCLUSIONS: The arginine metabolism balance is of vital importance during the pathogenesis and progression of chronic asthma. L-citrulline could be a powerful approach to restore airway NO production, potentially exhibiting therapeutic benefits among young patients with chronic asthma.

Quantitation of reduced IL-33 levels in human serum: mitigating interference from endogenous binding partners.

Aim: IL-33 is a potential therapeutic target but commercially available assays for the quantitation of systemic IL-33 have poor reliability. Results: In commercial IL-33 kits, interference from endogenous binding partners (e.g., soluble ST2) causes under-quantitation. Mitigating this required acid dissociation and addition of the detection reagent simultaneously with the capture step. This enabled detection of total, reduced (active) levels of IL-33 in human serum (LLOQ 6.25 pg/ml). Conclusion: Acid treatment of serum samples dissociates IL-33 from endogenous binding partners, increasing soluble ST2 tolerance to >1000 ng/ml. The modified method was specific for reduced endogenous IL-33. Analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no difference in serum IL-33.

Titanium Dioxide Nanoparticles Exacerbate Allergic Airway Inflammation via TXNIP Upregulation in a Mouse Model of Asthma.

Titanium dioxide nanoparticles (TiO2NPs) are widely used in industrial and medicinal fields and in various consumer products, and their increasing use has led to an increase in the number of toxicity studies; however, studies investigating the underlying toxicity mechanism have been rare. In this study, we evaluated potential toxic effects of TiO2NPs exposure on lungs as well as the development of asthma through the ovalbumin (OVA)-induced mouse model of asthma. Furthermore, we also investigated the associated toxic mechanism. TiO2NPs caused pulmonary toxicity by exacerbating the inflammatory response, indicated by an increase in the number and level of inflammatory cells and mediators, respectively. OVA-induced asthma exposed mice to TiO2NPs led to significant increases in inflammatory mediators, cytokines, and airway hyperresponsiveness compared with those in non-exposed asthmatic mice. This was also accompanied by increased inflammatory cell infiltration and mucus production in the lung tissues. Additionally, TiO2NPs decreased the expression of B-cell lymphoma 2 (Bcl2) and the expressions of thioredoxin-interacting protein (TXNIP), phospho-apoptosis signal-regulating kinase 1, Bcl2-associated X, and cleaved-caspase 3 were escalated in the lungs of asthmatic mice compared with those in non-exposed asthmatic mice. These responses were consistent with in vitro results obtained using human airway epithelial cells. TiO2NPs treated cells exhibited an increase in the mRNA and protein expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α with an elevation of TXNIP signaling compared to non-treated cells. Moreover, pathophysiological changes induced by TiO2NP treatment were significantly decreased by TXNIP knockdown in airway epithelial cells. Overall, TiO2NP exposure induced toxicological changes in the respiratory tract and exacerbated the development of asthma via activation of the TXNIP-apoptosis pathway. These results provide insights into the underlying mechanism of TiO2NP-mediated respiratory toxicity.

Poly-L-arginine promotes asthma angiogenesis through induction of FGFBP1 in airway epithelial cells via activation of the mTORC1-STAT3 pathway.

Angiogenesis is a key characteristic of asthma airway remodeling. By releasing cationic granule proteins, such as major basic protein (MBP), activated eosinophils play a prominent role in asthma, but the underlying mechanisms are still not fully understood. In this study, we demonstrated that fibroblast growth factor-binding protein 1 (FGFBP1) was dramatically upregulated in airway epithelial cell lines treated by poly-L-arginine (PLA), a mimic of MBP. Elevated FGFBP1 expression was also detected in asthma clinical samples, as well as in ovalbumin (OVA)-induced chronic asthma mouse models. PLA enhanced FGFBP1 expression through activation of the mechanistic target of rapamycin complex 1-signal transducer and activator of transcription 3 (mTORC1-STAT3) signaling pathway. STAT3 transactivated FGFBP1 by directly binding to the promoter of the FGFBP1 gene. Furthermore, we identified that FGFBP1 secreted by PLA-treated airway epithelial cells served as a proangiogenesis factor. Lastly, we found the mTORC1-STAT3-FGFBP1 signaling pathway was activated in an OVA-induced chronic asthma model with airway remodeling features. Rapamycin treatment alleviated respiratory symptoms and reduced angiogenesis in asthmatic mice. Therefore, activation of the mTORC1-STAT3-FGFBP1 pathway in the airway epithelium contributes to the progress of angiogenesis and should be targeted for the treatment of asthma.

Serum decoy receptor 3 (DcR3): a promising biomarker for atopic asthma in children.

Bronchial asthma is a common chronic inflammatory disease with high prevalence and morbidity, particularly in school-aged children. Decoy receptor 3 (DcR3) is a soluble decoy receptor that belongs to the tumor necrosis factor receptor superfamily and has been reported to be elevated in several allergic and inflammatory diseases. This study was designed to determine the role of DcR3 in pediatric asthma. The serum DcR3 levels were analyzed in 85 subjects (60 pediatric patients with bronchial asthma and 25 age- and sex-matched healthy control children) using the enzyme-linked immunosorbent assay technique. Patients with asthma had higher serum DcR3 levels than healthy control subjects (p = 0.007). In the atopic group of patients with asthma, the serum DcR3 levels were inversely correlated with the asthma control test score (R = - 0.392, p = 0.039). Overall, DcR3 could be a promising biomarker of atopic asthma, specifically in pediatric patients.

Evaluation of blood and tooth element status in asthma cases: a preliminary case-control study.

BACKGROUND: Asthma is a common respiratory disorder; some data were present on the correlation between increased levels of trace elements and the risk of asthma development. It was aimed to evaluate the levels of 13 selected blood and tooth elements (magnesium, phosphorus, calcium, chromium, manganese, iron, copper, zinc, strontium, molybdenum, cadmium, lead, mercury) in a well-controlled asthma group and the control group. METHODS: During the study period, 17 asthma patients and 26 age and gender-matched healthy children donated shed deciduous teeth having neither decay nor filling and enrolled for the study. The element levels in blood and teeth matrixes were analyzed with inductively coupled plasma mass spectrometry. Differences in blood and tooth elements in groups were evaluated with generalized linear models after adjusting confounding factors. RESULTS: After adjusting the child's "z scores of body mass index for age", history of iron deficiency anemia, and status of parental smoking, the generalized linear model revealed significantly lower tooth magnesium levels, lower blood zinc levels, and lower blood zinc/copper ratio in the asthma group than the control group (p = 0.042, p = 0.034, p = 0.002, respectively). Other studied elements for tooth and blood matrixes were similar in groups. CONCLUSION: Our study revealed some differences in tooth and blood element levels in the asthma group. Further studies on zinc and magnesium levels of severe asthma cases are necessary for the interpretation of the results.

Derangement of cell cycle markers in peripheral blood mononuclear cells of asthmatic patients as a reliable biomarker for asthma control.

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity.

FSTL1 aggravates OVA-induced inflammatory responses by activating the NLRP3/IL-1ß signaling pathway in mice and macrophages.

OBJECTIVE: Asthma, a well-known disease with high morbidity, is characterized by chronic airway inflammation. However, the allergic inflammation mechanisms of follistatin-like protein 1 (FSTL1) have not been elucidated. This study aims to investigate the effects of FSTL1 in ovalbumin (OVA)-induced mice and macrophages on nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3)/interleukin-1ß (IL-1ß) signaling pathway. METHODS: Mice were randomly divided into control-WT, OVA-WT, control-Fstl1±, OVA-Fstl1±. Histological changes were assessed by HE and PAS staining. The protein levels of Muc-5AC, FSTL1, NLRP3, and IL-1ß in lung tissue were detected by immunohistochemistry and ELISA. The bronchoalveolar lavage fluid (BALF) in mice and human serum samples were detected by ELISA. Then, mice were grouped into control, FSTL1, MCC950 + FSTL1 to further investigate the relationship between FSTL1 and NLRP3/IL-1ß. Alveolar macrophage cells (MH-S cells) were separated into control, OVA, FSTL1, OVA + FSTL1, OVA + siNC, OVA + siFSTL1, MCC950, and FSTL1 + MCC950 groups to explore the effect of FSTL1 on the NLRP3/IL-1ß signaling. The protein expression of NLRP3 and IL-1ß in MH-S cells was detected by Western blot analysis. RESULTS: The present results uncovered that Fstl1± significantly ameliorated OVA-induced Muc-5AC production and mucus hypersecretion. Fstl1± was also found to decrease the production of inflammatory cytokines and inflammatory cell infiltration in OVA-induced asthmatic mice. Meanwhile, the serum concentrations of FSTL1 and IL-1ß were higher in  asthma subjects than the health subjects, and Fstl1± ameliorated the production of NLRP3 and IL-1ß in OVA-induced asthmatic mice. Furthermore, mice by injected FSTL1 substantially stimulated the expression of NLRP3 and IL-1ß, while pretreatment with MCC950 in mice significantly weakened the production of NLRP3 and IL-1ß induced by injection FSTL1. Pretreatment with siFSTL1 or MCC950 significantly reduced the production of NLRP3 and IL-1ß induced by OVA or FSTL1 in MH-S cells. CONCLUSIONS: The study results showed that FSTL1 played an important role in allergic airway inflammation by activating NLRP3/IL-1ß. Hence, inhibition FSTL1 could be applied as a therapeutic agent against asthma.

The clinical significance of spondin 2 eccentric expression in peripheral blood mononuclear cells in bronchial asthma.

BACKGROUND: Bronchial asthma (BA) was a heterogeneous disease characterized by chronic airway inflammation. Spondin 2 (SPON2) was reported to be implicated in the integrin pathway, protein metabolism, and drug-induced lupus erythematosus. The purpose of this study was to evaluate the significance of SPON2 in BA diagnosis and treatment. METHODS: Peripheral blood samples were obtained from 137 BA pediatric patients (61 mild-to-moderate BA and 76 severe BA) and 59 healthy children. Subject's information, clinical indexes, pulmonary ventilation functions were recorded in the two groups. Peripheral blood mononuclear cells (PBMCs) were isolated from patients' samples. qRT-PCR and ELISA assays were employed to examine the levels of SPON2 and inflammatory cytokines, respectively. Pearson's correlation analysis confirmed the association between SPON2 and inflammatory cytokines. Receiver operating characteristic (ROC) analysis was used to evaluate the potentials of SPON2 in terms of BA detection and discriminating against the severity of BA. RESULTS: Bioinformatics analysis showed that SPON2, OLFM4, XIST, and TSIX were significantly upregulated, while KDM5D and RPS4Y1 were reduced in BA. GO analysis verified that these six genes were mainly involved in neutrophil degranulation, neutrophil activation involved in immune response, neutrophil activation, and neutrophil-mediated immunity. After isolating PBMCs, we found that SPON2 was remarkably increased in BA pediatric group compared with healthy children, and the relative levels of SPON2 were related to the severity of BA. The receiver operating characteristic (ROC) analysis revealed the high potentials of SPON2 in BA diagnosis (AUC was 0.8080) and severity distinctions (AUCs were 0.7341 and 0.8541, respectively). Also, we found that there were significant differences in fractional exhaled nitric oxide (FeNO), forced expiratory volume in 1 s (FEV1)%, FEV1/ forced vital capacity (FVC)%, immunoglobulin E (IgE), serum eosinophils, and serum neutrophils between mild-to-moderate BA group and severe BA group. Finally, SPON2 was negatively correlated with IL-12 while positively associated with IL-4, IL-13, and IL-17A. CONCLUSIONS: SPON2 was a viable biomarker for diagnosing and degree of severity in BA, providing more insight into exploring BA and treatment's pathogenesis.

In vitro and in vivo relaxation and anti-inflammation of natural flavonoids from Elaeagnus pungens leaf via L-type calcium channel and targeting MAPK signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), the leaf of Elaeagnus pungens Thunb. (Family Elaeagnaceae) is a herb documented as an antiasthmatic remedy to treat the severe asthma, bronchitis and other respiratory diseases in the early material medica "Bencao Gangmu" (Ming dynasty, about 442 years ago). AIM OF THE STUDY: This work is purposed to investigate the pharmacological effects and mechanism of total flavonoids from Elaeagnus pungens leaves (FLA) on asthma in vivo and vitro. MATERIALS AND METHODS: Female BALB/c mice were sensitized by intraperitoneal injection of OVA with aluminum hydroxide and intranasal challenged with OVA. After treatment with FLA (10, 20 mg/kg p.o.), the behaviors of mice were observed by score evaluation. Enumeration of total cells and OVA-specific IgE assay in the blood were measured as well as enumeration of total cells and cytokines assay in the BALF. Furthermore, histopathological analysis was performed by HE staining. The in vitro relaxing action on muscle force of FLA (0.0316-10.0 mg/ml) was evaluated using isometric tension in tracheal rings, and VDLCC currents were recorded to explore the relaxation mechanism in the isolated tracheal rings and mouse ASM cells, respectively. In vitro anti-inflammatory actions were assessed with LPS-stimulated RAW 264.7 macrophages. The production of inflammatory mediators and MAPK signaling pathway was estimated using ELISA and Western blotting analysis, respectively. RESULTS: The high dose of flavones from E. pungens leaf (20 mg/kg) can significantly improve the symptom of asthma breakout and relieve the lung swelling. FLA treatment decreased eosinophils and leukocytes numbers in blood and BLAF with a dosedependent manner. Furthermore, the inhibiting effect of FLA on the level of Ig E and inflammatory-related cytokines including TNF-α, IL-5 showed dose-independent. FLA relaxed high K + -induced contraction in a dose-dependent manner. The maximal relaxation produced by FLA was 99.7% (IC 50 = 0.46 mg/ml). The whole-cell VDLCC currents were abolished by FLA (3.16 mg/ml) and FLA significantly decreased the maximal amplitude of VDLCCs. No cytotoxic effect of FLA was observed in RAW264.7 cells under the tested concentrations (1-300 µg/mL). The increased IL-6 and NO by the stimulation of LPS in RAW264.7 cells were significantly inhibited by FLA in the dosedependent manner. Treatment with LPS in the presence of FLA, LPS-induced phosphorylation of ERK1/2 and JNK was inhibited in the macrophages. CONCLUSION: FLA from Elaeagnus pungens leaf can alleviate the inflammation symptom via reducing the eosinophils and leukocytes numbers as well as the production of pro-inflammatory cytokines. This anti-inflammatory effect is related to the modulation of the MAPK signaling pathway. FLA can relax the precontracted TRs by blocking the VDLCCs, which interrupts extracellular Ca 2+ influx and inhibit the rise of [Ca 2+ ]i. It strongly suggests that these flavonoids components are the substances basis of Elaeagnus pungens leaves for allergic action, bronchospasm and inflammation in asthma.