MicroRNA Levels in Exhaled Breath Condensate of Patients with Esophageal Atresia.

MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the regulation of inflammation and have not been evaluated in exhaled breath condensates (EBC) of patients with esophageal atresia and tracheoesophageal fistula (EA-TEF). It is aimed to evaluate the levels of miRNA-21 and miRNA-24 in EBC of patients with EA-TEF. Patients who received surgery for EA-TEF (EA) were assessed for age, sex, types of anomaly, surgical treatments, and respiratory problems. A 500-1000 mL of EBC was obtained from each participant with EcoScreen. The levels of miRNA-21 and miRNA-24 in the EBC were analyzed by real-time polymerase chain reaction and compared between the EA group and the control group consisting of healthy children with no history of respiratory problems (n = 17). The levels of miRNAs in relation to respiratory problems and gastroesophageal reflux (GER) were also assessed. A total of 19 patients were enrolled in the EA group with a mean age of 7.8 ± 3.2 years and a male-to-female ratio of 10:9 EA cases had significantly lower levels of miRNA-21 (P < 0.05) compared to that in control group. The miRNA-24 levels did not differ between groups (P > 0.05). EA patients with positive pH testing for GER (n = 6) and fundoplication (n = 6) had higher levels of miRNA-21 than those with normal pH testing and without fundoplication, respectively (n = 13, P < 0.05). The levels of miRNA-21 and miRNA-24 did not differ between patients with and without proton pump inhibitor treatment (P > 0.05). The lower levels of miRNA-21 in the EBC of EA patients suggest a hyperreactive airway problem, which may be associated with GER and its surgical treatment.

Low indoleamine 2,3-dioxygenase activity in persistent food allergy in children.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan (Trp) to kynurenine (Kyn), has been demonstrated to contribute to modulation of allergic responses. However, the role of IDO in food allergy has not yet been elucidated. METHODS: Serum Trp and Kyn concentrations were analyzed by high-pressure liquid chromatography. Expression of IDO gene was measured by real-time PCR. The levels of interleukin (IL)-4, IL-10, and interferon (IFN)-γ in cell culture supernatants were measured by ELISA. RESULTS: Kyn/Trp (IDO activity) was significantly lower in subjects with food allergy (n = 100) than in aged-matched healthy controls (n = 112) (P = 0.004). Kyn/Trp was decreased from healthy through completely tolerant, partially tolerant, and reactive ones [LN transformation (mean ± SEM) healthy: 3.9 ± 0.02 µM/mM; completely tolerant: 3.83 ± 0.04; partially tolerant: 3.8 ± 0.06; reactive: 3.7 ± 0.04] (P = 0.008). The frequency of genetic polymorphisms of IDO did not reveal a significant association with Trp, Kyn, and Kyn/Trp in healthy and food-allergic cases. Culture of PBMC experiments yielded that IDO mRNA expression was not different between tolerant and reactive groups. IL-4 synthesis when stimulated with casein increased significantly in subjects who are reactive and tolerant to foods (P = 0.042, P = 0.006, respectively). Increase in IL-10 synthesis was observed only in children tolerant to milk, but not in reactive ones. IFN-γ synthesis, when stimulated with IL-2 and ß-lactoglobulin in cell culture, was significantly higher in subjects tolerant to milk than in the reactive ones (P = 0.005 and P = 0.029, respectively). CONCLUSION: Our results imply the probability of involvement of IDO in development of tolerance process, and we presume that high IDO activity is associated with nonresponsiveness to food allergens despite allergen sensitization.

Mechanisms of peripheral tolerance to allergens.

The immune system is regulated to protect the host from exaggerated stimulatory signals establishing a state of tolerance in healthy individuals. The disequilibrium in immune regulatory vs effector mechanisms results in allergic or autoimmune disorders in genetically predisposed subjects under certain environmental conditions. As demonstrated in allergen-specific immunotherapy and in the healthy immune response to high-dose allergen exposure models in humans, T regulatory cells are essential in the suppression of Th2-mediated inflammation, maintenance of immune tolerance, induction of the two suppressive cytokines interleukin-10 and transforming growth factor-ß, inhibition of allergen-specific IgE, and enhancement of IgG4 and IgA. Also, suppression of dendritic cells, mast cells, and eosinophils contributes to the construction of peripheral tolerance to allergens. This review focuses on mechanisms of peripheral tolerance to allergens with special emphasis on recent developments in the area of immune regulation.

Importance of serum basal tryptase levels in children with insect venom allergy.

BACKGROUND: The importance of serum basal tryptase (sBT) levels on patients with venom allergy is highlighted in recent adulthood studies. The aim of this study was to evaluate the sBT levels of venom-allergic children with varying severity of clinical reactions. We also aimed to document the association between sBT levels and severe systemic reactions (SR). METHODS: Serum basal tryptase levels were estimated by UniCAP (Pharmacia & Upjohn, Uppsala, Sweden). Children who suffered from large local reaction (LLR) or SR after insect stings were included along with healthy control subjects without a history of any local or SR after insect stings. RESULTS: A total of 128 children (55 with SR, 18 with LLR, and 55 age and sex-matched control subjects) with a median age of 8.9 years (range 3.2-17.4) were enrolled. Severe SR was encountered in 24 (44%) patients with SRs. The median level of sBT in children with SRs (median, interquartile range) [4.2 µg/l (3.6-4.9)] was significantly higher than in children with LLRs [3.1 µg/l (2.5-4.0)] and healthy control subjects [2.9 µg/l (2.3-3.4)] (P < 0.001). Logistic regression analysis revealed sBT ≥ 4.8 µg/l as a significant risk factor for severe SR (5.7 [1.5-21.4]; P = 0.01) in children with venom allergy. CONCLUSIONS: Our results indicate that sBT levels are associated with a higher risk of severe SR in children with insect venom hypersensitivity. Determination of sBT levels may help clinicians to identify patients under risk of severe SRs and optimal and timely use of therapeutic interventions in children with venom allergy.

The reliability and validity of Turkish version of Childhood Asthma Control Test.

INTRODUCTION: The reliability and validity of Turkish version of Childhood Asthma Control Test (C-ACT). PURPOSE: The management of asthma is an important as well as difficult issue of physician's daily practice particularly in busy clinical settings. C-ACT was created to identify asthma control levels in children aged 4-11 years. Our aim was to evaluate the reliability, validity and responsiveness of C-ACT in a Turkish sample of children with asthma. METHOD: In this multicenter study, 368 children were enrolled. C-ACT was completed every month by parents and patients who were evaluated in 3 visits within 2 month intervals. At each visit, physicians interpret the control level and decided for the treatment step as established in GINA guidelines. RESULTS: The internal consistency reliability of the Turkish version of C-ACT (C-ACT1 to C-ACT5) was found to be 0.82, 0.83, 0.82, 0.82 and 0.80, respectively (reliability statistics, Cronbach's alpha). Test-retest reliability was 0.71. There was significant correlation between C-ACT and physician's assessment of asthma control at visit 1 (r = 0.65, P < 0.001). CONCLUSIONS: Turkish version of C-ACT is an accurate and reliable tool to evaluate asthma control in children aged 4-11 years. Its widespread use may facilitate appropriate assessment of asthma control and may lead to decrease the number of uncontrolled patients.

The effect of CD14 C159T polymorphism on in vitro IgE synthesis and cytokine production by PBMC from children with asthma.

BACKGROUND: Even though the genotype at the promoter region of the CD14 molecule is known to affect the atopic phenotypes, the cellular and molecular basis of this association is largely unknown. OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS) on IgE production and cytokine profile by peripheral blood mononuclear cells (PBMC) obtained from asthmatic children with the TT and the CC genotypes at position -159 of the CD14 gene. METHODS: Peripheral blood mononuclear cells from asthmatic children with alternative genotypes at CD14 C159T locus were stimulated with 2 and 200 ng/ml LPS in vitro. The IgE, IgG and, IgM response was determined by ELISA and Ig έ-germline, IgG, and IgM transcription by real-time PCR. A cluster of cytokines was measured by cytometric bead array. RESULTS: Asthmatic children with the TT genotype but not those with the CC genotype responded with increased IgE synthesis and germline transcription to LPS stimulation. There were no genotype-related differences in IgG and IgM. TT but not the CC genotype was associated with significantly increased interleukin (IL)-4/IL-12 and IL-4/interferon-gamma (IFN-γ) ratios in the culture supernatant. There were no genotype-related differences in IL-1ß, IL-7, IL-10, IL-13, IL-17A, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein, and tumor necrosis factor alpha. CONCLUSION: Peripheral blood mononuclear cells from asthmatic children with the TT genotype at position -159 of the CD14 gene make more IgE than those with the CC genotype following LPS stimulation because of increased germline transcription and have an augmented Th2 cytokine profile.

Eccrine sweat response in children with asthma.

BACKGROUND: Even though there is a general conviction among parents of asthmatic children and pediatricians that asthmatic children sweat more than healthy ones, this has not been formally tested. AIM: To determine sweating response and factors affecting this response in children with asthma and compare these findings with healthy children. METHOD: Eighty-two children with asthma and 51 healthy controls aged 6-18 years were enrolled in the study. Transepidermal water loss (TEWL) was measured on palmar, volar, forehead, and back surfaces before and after exercise and was expressed as the difference between the measurements recorded before and after exercise. RESULTS: Transepidermal water loss measurements (after exercise - resting) on the palmar surface were higher in children with asthma [22.8 g/m(2 )h (15-34.3)] compared with healthy children [15.2 g/m(2) h (6-22.2)] (P < 0.001). However, a gender stratified analysis showed that the TEWL measurements were higher on all surfaces only in boys but not in girls. Within the group of asthmatic children, TEWL measurements on the volar surface and back were lower in patients using anti-inflammatory therapy compared with those who were on as needed bronchodilator therapy only. CONCLUSION: Our results show that asthma is associated with a higher rate of sweating response to exercise in boys, and anti-inflammatory treatment decreases the amount of sweating. The relationship of eccrine sweating with muscarinic receptor response and methacholine hyperresponsiveness remains to be determined.

A multicenter survey of childhood asthma in Turkey. II: Utilization of asthma drugs, control levels and their determinants.

Many surveys worldwide have consistently demonstrated a low level of asthma control and under-utilization of preventive asthma drugs. However, these studies have been frequently criticized for using population-based samples, which include many patients with no or irregular follow-ups. Our aim, in this study, was to define the extent of asthma drug utilization, control levels, and their determinants among children with asthma attending to pediatric asthma centers in Turkey. Asthmatic children (age range: 6-18 yr) with at least 1-yr follow-up seen at 12 asthma outpatient clinics during a 1-month period with scheduled or unscheduled visits were included and were surveyed with a questionnaire-guided interview. Files from the previous year were evaluated retrospectively to document control levels and their determinants. From 618 children allocated, most were mild asthmatics (85.6%). Almost 30% and 15% of children reported current use of emergency service and hospitalization, respectively; and 51.4% and 53.1% of children with persistent and intermittent disease, respectively, were on daily preventive therapy, including inhaled corticosteroids. Disease severity [odds ratio: 12.6 (95% confidence intervals: 5.3-29.8)], hospitalization within the last year [3.4 (1.4-8.2)], no use of inhaled steroids [2.9 (1.1- 7.3)], and female gender [2.3 (1.1-5.4)] were major predictors of poor asthma control as defined by their physicians. In this national pediatric asthma study, we found a low level of disease control and discrepancies between preventive drug usage and disease severity, which shows that the expectations of guidelines have not been met even in facilitated centers, thus indicating the need to revise the severity-based approach of asthma guidelines. Efforts to implement the control-based approach of new guidelines (Global Initiative for Asthma 2006) would be worthwhile.

Oxidative stress and its determinants in the airways of children with asthma.

BACKGROUND: There is ample evidence for the existence of a systemic oxidative stress in childhood asthma but relatively little information on the oxidant stress in the airways. OBJECTIVE: To determine the extent of oxidant/antioxidant imbalance and describe its determinants in the airways of asthmatic children including asthma severity and the genotype of the antioxidant enzymes. METHODS: One hundred and ten children with mild asthma, 30 children with moderate asthma and 191 healthy controls were included in the study. Exhaled breath condensate (EBC) was collected from all children with EcoScreen. Levels of malondialdehyde were measured as the indicator of oxidative stress, and of reduced glutathione as the indicator of antioxidant defense. Children were genotyped for the presence of null variants of glutathione S transferase (GST) T1 and GSTM1, and ile105val variant of GSTP1. Risk factors were analyzed with multivariate logistic regression. RESULTS: EBC contained significantly higher levels of malondialdehyde and lower levels of reduced glutathione in asthmatic children compared with healthy controls (P < 0.001 for each), whereas there was no difference between mild and moderate asthmatics. Multivariate logistic regression identified asthma as the only independent factor contributing to oxidative stress. Genotypes of the antioxidant enzymes had no effect on the oxidative burden. CONCLUSIONS: Asthma is associated with an extremely powerful oxidative stress not only in the systemic circulation but also in the airways.

Turkish physicians' perception of allergic rhinitis and its impact on asthma.

BACKGROUND: The clinical association of rhinitis and asthma has been recognized for centuries, leading to a current definition of 'one airway, one disease'. Current findings indicate that the optimal treatment of rhinitis might improve coexisting asthma. OBJECTIVES: To evaluate perception of the asthma-rhinitis association and to determine their capability in managing rhinitis. METHODS: Participants of three national asthma meetings, in 2004, were asked to complete a questionnaire which, aside from demographic queries, elicited responses in order to reflect agreement or not with the given statements. RESULTS: Of the 354 attendees, 220 (62%) responded to the questionnaire. Although over 80% reported good perception of the asthma-rhinitis association, only one-third were examining nasal mucosa of asthmatics, and over 70% believed sinus X-ray, blood eosinophil count, and determination of total serum IgE were required to diagnose allergic rhinitis. Between 20% and 40% of the physicians reported misuse of medications for rhinitis. CONCLUSIONS: This survey documented that the asthma-rhinitis association is generally recognized by physicians treating asthma in Turkey. However, there are certain training needs in terms of attitude towards examinations and appropriate use of laboratories and medications for rhinitis. Improved understanding/management of rhinitis may contribute to asthma care as well as patient satisfaction and adherence to treatment.

Comparison of two methods for exhaled breath condensate collection.

BACKGROUND: Exhaled breath condensate (EBC) is a noninvasive method to obtain samples from fluids lining the respiratory surfaces. Even though various methods and devices are now available, the relative efficiency of these methods for recovering airway mediators and characterizing EBC has not been established. AIM: To compare the volume, pH, lipid mediator, and protein concentrations in EBCs collected by two commonly used commercially available devices, RTube and ECoScreen. METHODS: Exhaled breath condensates were obtained consecutively using the RTube and ECoScreen methods from 30 healthy, nonallergic adults. Samples were immediately placed on dry ice after collection. pH was measured after argon deaeration. Cysteinyl leukotrienes (cys-LTs) were measured as a representative lipid mediator and eotaxin as the protein mediator by using enzyme-linked immunosorbent assay. RESULTS: The mean volume of samples obtained with ECoScreen (1880 +/- 116 microl) was significantly higher than that obtained with RTube (1405 +/- 82 microl) (P < 0.001). Concentrations of both cys-LTs [205.4 pg/ml (65.5-472.3) with ECoScreen vs 21.6 (11.87-152.2) with RTube, P < 0.001] and eotaxin [17.0 pg/ml (11.4-22.4) with ECoScreen vs 11.7 (10.5-13.5) with RTube, P = 0.01] were significantly higher in samples collected with ECoScreen than with RTube. There was no significant difference between the pH measurements. CONCLUSION: Compared with RTube, collection of exhaled breath by ECoScreen allows larger volumes to be collected and detects protein and lipid mediators with greater sensitivity. These differences in mediator recovery may be due to the differences in the collection temperature.

ALOX5 promoter genotype, asthma severity and LTC production by eosinophils.

BACKGROUND: The number of Sp1-Egr1 binding tandem repeats at the ALOX5 promoter influences gene transcription and may modify the response to anti-leukotriene treatment. The relationship of ALOX5 variants to asthma severity and leukotriene production by eosinophils is unknown. OBJECTIVE: To characterize ALOX5 mRNA expression and cysteinyl-leukotriene production by eosinophils from individuals bearing ALOX5 promoter deletional variants and their association with the severity of childhood asthma. METHODS: Eosinophils from adult asthmatics bearing only variant alleles (with other than five tandem repeats on both chromosomes, non5/non5) or no variant alleles (5/5) were cultured in vitro and ALOX5 expression and leukotriene secretion were measured. A total of 621 children with mild or moderate-severe asthma were genotyped at the ALOX5 core promoter. RESULTS: Asthmatics with non5/non5 genotype expressed less ALOX5 mRNA and produced less LTC4 into culture supernatants than 5/5 individuals (6.4 +/- 2.0 and 20.0 +/- 5.0 pg/ml, n = 5; P < 0.05). More asthmatic children bearing non5/non5 genotype had moderate-severe asthma than children with the 5/5 genotype (5.3% vs. 1.4%, P = 0.008). Multivariate logistic regression identified ALOX5 promoter genotype as a significant predictor of disease severity (OR = 3.647, 95% CI: 1.146-11.608, P = 0.03). Consistent with these findings, children bearing the non5/non5 genotype had greater bronchomotor response to exercise as measured by the maximum fall after exercise and the area under the exercise curve (P < 0.05 for both). CONCLUSION: Our results suggest that children who express the asthma phenotype despite having a genetic variant that impairs their ability to express ALOX5 have more severe disease and thus are more likely to have asthma symptoms.

The effect of polymorphisms at the CD14 promoter and the TLR4 gene on asthma phenotypes in Turkish children with asthma.

BACKGROUND: Endotoxin, with its potential to enhance type 1 immunity, is a significant player in the hygiene hypothesis. The combined effects of the genetic variants of various molecules in the endotoxin response pathway on asthma related phenotypes are largely unknown. OBJECTIVE: To investigate the effects of the genetic variants of CD14 and TLR4 genes on asthma phenotypes in a large number of asthmatic children. METHODS: 613 asthmatic children were genotyped at the CD14-C159T, TLR4-A896G and TLR4-C1196T loci. IgE, eosinophil numbers and FEV1 were compared in 327 children who were not on any controller medications and were symptom free. Multivariate logistic regression was used to determine the factors associated with total IgE. RESULTS: Among children with atopic asthma, total IgE levels were significantly different among the three genotypes in the co-dominant model [CC: 435 kU/l (interquartile range: 146-820); CT: 361 (140-710); TT 204 (98-435), P = 0.035]. TT genotype was significantly and independently associated with lower IgE levels (OR: 0.5 95%; CI = 0.28-0.90, P = 0.021). Both TLR4-A896G and TLR4-C1196T polymorphisms were more frequent in the mild asthma group with atopy (P = 0.032, 0.018, respectively). The combined effects of the genetic variants in CD14 and TLR4 genes did not improve the observed associations. CONCLUSION: Our study demonstrates that the CD14-C159T promoter variant influences total IgE levels and also indicates that the T allele has a more profound effect on total IgE in children with atopic asthma. Polymorphisms in the TLR4 gene may be associated with milder forms of disease in atopic asthmatics in the population studied.