Oxidative stress stimulation leads to cell-specific oxidant and antioxidant responses in airway resident and inflammatory cells.

AIMS: The imbalance between reactive oxygen species (ROS) and the antioxidant response has been linked to various airway diseases, including asthma. However, knowledge on cell-specific responses of the airway resident and inflammatory cells against increased oxidant stress is very limited. We aim to better understand the cell-specific antioxidant response that contributes to the pathophysiology of lung disease in response to oxidative stress. MATERIALS AND METHODS: The human cell lines of epithelial, fibroblast, endothelial, monocyte, eosinophil and neutrophil were incubated with tert-butyl hydroperoxide (tBHP) or cigarette smoke condensate (CSC). Following stimulation, cell viability, total oxidant and antioxidant activity were assessed in both residential and inflammatory cells. Human Oxidative Stress Plus RT2 Profiler PCR array was used to determine 84 gene expression differences in oxidant and antioxidant pathways following oxidant stimulus in all cells. KEY FINDINGS: We showed that various cell types respond differently to oxidative stress inducers, with distinct gene expression and oxidant-antioxidant generation. Most importantly, eosinophils increased the activity of all main antioxidant enzymes in response to both oxidants. Monocytes, on the other hand, showed no change in response to each stimulation, whereas neutrophils only increased their CAT activity in response to both stimuli. The increase in NRF2-regulated genes HSPA1A, HMOX1 and DUSP1 after both tBHP and CSC in epithelial cells and fibroblasts indicates Nfr2 pathway activation. SIGNIFICANCE: This study advances our knowledge of the molecular and cellular mechanisms of cell-specific antioxidant response upon exposure to oxidative stress. Additionally, our observations imply that the eosinophils' distinct biological response may be utilized for endotype-based cell-targeted antioxidant therapy.

The Expression Profile of Protease Inhibitors in the Airway Epithelial Cells after Allergen (Der p 1) Stimulation.

BACKGROUND: Airway epithelial cells are constantly exposed to intracellular and extracellular proteases that play a pivotal role in several airway diseases. Dermatophagoides pteronyssinus (Der p) 1 derived from house dust mite has protease activity that causes epithelial barrier defect and inflammatory response. Protease inhibitors released against proteases are involved in the maintenance of homeostasis. A disruption of the balance between proteases and protease inhibitors can lead to distortion of the cellular structures and cellular activities and thus culminate in disease processes. Although the effects of Der p 1 allergen on epithelial barrier integrity and inflammatory response are well-established, its contribution to protease inhibitor production is highly limited. OBJECTIVE: This study aimed to determine the profile of the protease inhibitor response to Der p 1 allergen in human airway epithelial cells, A549 and BEAS-2B. METHODS: Differentiated cells by the air-liquid interface were exposed to Der p 1 with or without Th2 type cytokines (IL-4 and IL-13). Gene expression of protease inhibitors was determined by qPCR at 2 different time points. RESULTS: We found that the effect of allergen exposure on the protease inhibitor profile can vary depending on the antigen concentration, treatment duration, and the presence or absence of type 2 cytokines. Gene expressions of serine protease inhibitor (SERPIN)B3 and SERPINB4 were increased following Th2 cytokine stimulation in both cell types at both time points, whereas SERPINB2 and TFPI-2 expressions were induced by 24-h Der p 1 stimulation in both cells. CONCLUSIONS: Our study suggests that Der p 1 exposure of the airway epithelium may have consequences related to its protease activity in the presence as well as in the absence of Th2 cytokines in the microenvironment.

House dust mite-derived allergens effect on matrix metalloproteases in airway epithelial cells.

Aim of the Study: Many allergens have protease activities. Although the immunomodulatory effects of these antigens are well known, the effects attributed to their protease activities are not thoroughly investigated. We set out to determine the effects of house dust mite (HDM) allergens with varying protease activities on bronchial epithelial cell functions. Materials and methods: BEAS-2B cells were maintained in ALI-culture and stimulated with Der p1 (cysteine protease), Der p6 (serine protease), and Der p2 (non-protease) with and without specific protease inhibitors or heat denaturation. Cell viability and epithelial permeability were measured with MTT and paracellular flux assay, respectively. The effect of heat denaturation on allergen structure was examined using in silico models. Matrix metalloproteinases (MMPs) were investigated at the transcription (qPCR), protein (ELISA), and functional (zymography) levels. Results: Epithelial permeability increased only after Der p6 but not after Der p1 or Der p2 stimulation. Der p2 increased both MMP-2 and MMP-9 expression, while Der p1 increased only MMP-9 expression. The heat-denatured form of Der p1 unexpectedly increased MMP-9 gene expression, which, through the use of in silico models, was attributed to its ability to change receptor connections by the formation of new electrostatic and hydrogen bonds. IL-8 and GM-CSF production were increased after Der p1 and Der p2 but decreased after Der p6 stimulation. IL-6 decreased after Der p1 but increased following stimulation with Der p6 and heat-denatured Der p2. Conclusion: Allergens in house dust mites are capable of inducing various changes in the epithelial cell functions by virtue of their protease activities.

The Montelukast Therapy in Asthmatic Children with and without Food Allergy: Does It Make Any Difference?

INTRODUCTION: Children with food allergy are at increased risk for asthma and asthma morbidity. Since leukotrienes are implicated in the pathogenesis of both asthma and probably in food allergies, we hypothesized that asthmatic children with concomitant food allergy may have a favorable response to antileukotriene treatment. METHODS: Asthmatic children aged 6-18 years with and without food allergy were treated with montelukast and placebo in a double-blind, placebo-controlled cross-over parallel-group study. The primary outcome of the study was improvement in FEV1%. Asthma control tests, spirometry and methacholine challenges were performed as well as Fractional Exhaled Nitric Oxide (FeNO) levels. PGD2, CystLT, and lipoxin levels were measured in exhaled breath condensate (EBC). RESULTS: A total of 113 children were enrolled and 87 completed the study in accordance with the protocol. At baseline, children with food allergy and asthma (FAA) had higher levels of PGD2 and CysLT levels in the EBC than children with asthma alone (AA) (p < 0.001 for each). In the montelukast arm, although FEV1% was significantly higher in the FAA group compared to AA (p = 0.005), this effect was linked to the baseline difference of FEV1% between both arms. Montelukast treatment failed to improve FEV1% in both groups compared to the placebo. No effect of montelukast was observed in the remaining study parameters. CONCLUSION: Although children with FAA do not show a more favorable response to montelukast treatment compared to AA, a significant difference between baseline PGD2 and CystLT levels between FAA and AA groups may point to a different endotype of childhood asthma.

Four subtypes of childhood allergic rhinitis identified by latent class analysis.

BACKGROUND: Childhood allergic rhinitis (AR) is clinically heterogenous. We aimed to identify distinct phenotypes among children with AR using data-driven techniques and to ascertain their association with patterns of symptoms, allergic sensitization, and comorbidities. METHODS: We recruited 510 children with physician-diagnosed AR, of whom 205 (40%) had asthma. Latent class analysis (LCA) was performed to identify latent structure within the data set using 17 variables (allergic conjunctivitis, eczema, asthma, family history of asthma, family history of allergic rhinitis, skin sensitization to 8 common allergens, tonsillectomy, adenoidectomy). RESULTS: A four-class solution was selected as the optimal model based on statistical fit. We labeled latent classes as: (1) AR with grass mono-sensitization and conjunctivitis (n = 361, 70.8%); (2) AR with house dust mite sensitization and asthma (n = 75, 14.7%); (3) AR with pet and grass polysensitization and conjunctivitis (n = 35, 6.9%); and (4) AR among children with tonsils and adenoids removed (n = 39, 7.6%). Perennial AR was significantly more common among children in Class 2 (OR 5.83, 95% CI 3.42-9.94, p < .001) and Class 3 (OR 2.88, 95% CI 1.36-6.13, p = .006). Mild and intermittent AR symptoms were significantly more common in children in Class 2 compared to those in Class 1. AR was more severe in Class 1 compared to other 3 classes, indicating that upper respiratory symptoms are more severe among children with isolated seasonal rhinitis, than in those with rhinitis and coexisting asthma. CONCLUSION: We have identified 4 phenotypes in school-age children with AR, which were associated with different patterns of clinical symptoms and comorbidities.

The effect of montelukast on early-life wheezing: A randomized, double-blinded placebo-controlled study.

BACKGROUND: Cysteinyl-leukotrienes are increased in the airways of infants with virus-associated wheezing. We aimed to determine the effects of a cysteinyl-leukotriene-1 receptor antagonist on symptoms during an early-life wheezing illness and to investigate the factors that affect the response to this drug. METHOD: This placebo-controlled double-blinded randomized controlled trial recruited children aged 3-36 months with wheezing illness and randomized to active drug or placebo for 56 days. A symptom score diary (SSD) was kept by the children's caregivers. RESULTS: One-hundred patients completed the study, and 62 (30 montelukast and 32 placebo) were analyzed. There were no significant differences in the percent of symptom-free days, symptom scores, and the need for rescue salbutamol between the two groups. However, the percent of symptom-free days within the first week was significantly higher for the montelukast than for the placebo group (13.8 ± 4.1% vs. 5.4 ± 3.4%; P = 0.028); wheezing score at 7th day was significantly lower for the montelukast than for the placebo group (0.5 ± 0.1 vs. 1.4 ± 0.2; P = 0.002). In addition, the number of inhaled ß2 -agonist rescue episodes per day during the first week was significantly lower for the montelukast compared with the placebo group (12.7 ± 1.8 vs. 19.2 ± 1.6; P = 0.013). Conclusions Our results indicate that montelukast may be effective for reducing caregiver-observed wheezing and the need for salbutamol during the first week of treatment for early-life wheezing. The impact for caregivers and the optimal duration of treatment will need to be explored in studies of larger size.

Doublesex and mab-3 related transcription factor 1 (DMRT1) is a sex-specific genetic determinant of childhood-onset asthma and is expressed in testis and macrophages.

BACKGROUND: Asthma is a disease affecting more boys than girls in childhood and more women than men in adulthood. The mechanisms behind these sex-specific differences are not yet understood. OBJECTIVE: We analyzed whether and how genetic factors contribute to sex-specific predisposition to childhood-onset asthma. METHODS: Interactions between sex and polymorphisms on childhood asthma risk were evaluated in the Multicentre Asthma Genetics in Childhood Study (MAGICS)/Phase II International Study of Asthma and Allergies in Childhood (ISAAC II) population on a genome-wide level, and findings were validated in independent populations. Genetic fine mapping of sex-specific asthma association signals was performed, and putatively causal polymorphisms were characterized in vitro by using electrophoretic mobility shift and luciferase activity assays. Gene and protein expression of the identified gene doublesex and mab-3 related transcription factor 1 (DMRT1) were measured in different human tissues by using quantitative real-time PCR and immunohistochemistry. RESULTS: Polymorphisms in the testis-associated gene DMRT1 displayed interactions with sex on asthma status in a population of primarily clinically defined asthmatic children and nonasthmatic control subjects (lowest P = 5.21 × 10(-6)). Replication of this interaction was successful in 2 childhood populations clinically assessed for asthma but showed heterogeneous results in other population-based samples. Polymorphism rs3812523 located in the putative DMRT1 promoter was associated with allele-specific changes in transcription factor binding and promoter activity in vitro. DMRT1 expression was observed not only in the testis but also in lung macrophages. CONCLUSION: DMRT1 might influence sex-specific patterns of childhood asthma, and its expression in testis tissue and lung macrophages suggests a potential involvement in hormone or immune cell regulation.

Pure endobronchial inflammatory myofibroblastic tumor in children.

Isolated endobronchial inflammatory myofibroblastic tumor is an unusual diagnosis among endobronchial masses in childhood. The presenting signs and symptoms may mimic asthma. Rigid bronchoscopy is effective for the diagnosis and treatment. Follow-up is mandatory to check for recurrent disease. Here in, the authors report on a 9-year-old girl with endobronchial inflammatory myofibroblastic tumor to emphasize the possibility of endobronchial lesion in children with longstanding obstructive symptoms.

Pepsin levels and oxidative stress markers in exhaled breath condensate of patients with gastroesophageal reflux disease.

AIM: To evaluate the pepsin and oxidative stress markers in exhaled breath condensate (EBC) in patients with gastroesophageal reflux disease (GERD). PATIENTS AND METHOD: Patients with a presumptive diagnosis of GERD with recurrent respiratory and gastrointestinal problems aged between 2 and 14 years were included in the study. All patients underwent pH monitoring. Patients with a reflux index (RI) ≥4 were assessed as the reflux group, and those with an RO <4 were assessed as the non-reflux group. Pepsin levels and oxidative stress markers [NO metabolites (NOX) and total sulphydrile (TSH) levels] were measured in the EBC. RESULTS: There were 24 patients in the reflux group [RI 17.6 (6.6-46.4)] [median, interquartile range] and 23 in the non-reflux group [RI 0.8 (0.5-1.9) (p<0.001). Pepsin levels in the EBC were below the level of detection. The median levels of NOx in the EBC of children with reflux [13.7 µmol/L (7.3-24.5)] were lower in than non-reflux group [21.0 µmol/L (14.0-25.2)] (p=0.034). There was a negative correlation between reflux index and NOX levels in EBC (rs: -0.331, p=0.023). In contrast, there was no difference in TSH levels between the reflux and non-reflux groups [37.4 µmol/L (30.2-44.6) vs 40.1 µmol/L (37.4-44.9), respectively, (p>0.05)]. CONCLUSION: Decreased levels of NOX in patients with GER disease suggest increased oxidative stress in airways of these patients.

The role of SCCA1 in asthma related physiological events in the airway epithelium and the effect of promoter variants on asthma and gene function.

BACKGROUND: Even though the systemic level of SCCA1, a serine protease inhibitor, was shown to be elevated in asthma, its physiological role is unknown. OBJECTIVE: We sought to determine the effect of SCCA1 on apoptosis, cytokine expression and mucus production by A549 cells and define the effect of promoter variants on gene expression and association with asthma. METHODS: SCCA levels were measured by ELISA. Promoter variants were determined by direct sequencing. 442 asthmatic children and 191 controls were genotyped by RFLP. The functional effect of the polymorphisms was assessed in transient transfection experiments using reporter constructs. A transcription factor ELISA was used for differential binding of GATA proteins to the variant region. The effects of SCCA1 on cytokine synthesis, mucus production and apoptosis were determined in A549 cells transfected with SCCA1 pcDNA vector. MUC5AC expression in A549 cells was determined with RT-PCR. RESULTS: SCCA1 protein level was significantly higher in asthmatic children compared to healthy controls. Four polymorphisms SCCA1 promoter that were in linkage disequilibrium were associated with skin test positivity in asthmatic children and showed higher promoter activity and higher binding of GATA-2 and GATA-3 after IL-4 + IL-13 stimulation. IL-6, IL-8 levels were significantly higher in cells transfected with SCCA1 whereas RANTES increased only after IL-4 stimulation. Transfection of A549 cells with SCCA1 resulted in decreased MUC5AC expression and conferred protection against apoptosis. CONCLUSION: Our results showed that SCCA1 has diverse effects on many of the cellular events that characterize asthma and its role extends beyond protease inhibition.