Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

BACKGROUND: Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. OBJECTIVES: To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. METHODS: Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; RT ) and permeability to fluorescent dextran. RESULTS: Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. CONCLUSION: This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma.

Impaired airway epithelial cell responses from children with asthma to rhinoviral infection.

BACKGROUND: The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood. OBJECTIVE: To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection. METHODS: Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-ß production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured. RESULTS: Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-ß production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-ß restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production. CONCLUSIONS: Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children and is only partially reversed by exogenous IFN-ß.

Expiratory flow limitation and breathing strategies in overweight adolescents during submaximal exercise.

OBJECTIVE: To investigate whether ventilatory factors limit exercise in overweight and obese children during a 6-min step test and to compare ventilatory responses during this test with those of healthy weight children. DESIGN: Cross-sectional, prospective comparative study. SUBJECTS: Twenty-six overweight/obese subjects and 25 healthy weight subjects with no known respiratory illness. MEASUREMENTS: Various fatness and fat distribution parameters (using air displacement plethysmography and anthropometry), pulmonary function tests, breath-by-breath gas analysis during exercise, perceived exertion. RESULTS: Young people who are overweight or obese are more likely to experience expiratory flow limitation (expFL) during submaximal exercise compared with their healthy weight peers [OR 7.2 (1.4, 37.3), P=0.019]. Subjects who had lower lung volumes at rest were even more likely to experience exercise-induced expFLs [OR 8.35 (1.4-49.3)]. Both groups displayed similar breathing strategies during submaximal exercise. CONCLUSION: Young people who are overweight/obese are more likely to display expFL during submaximal exercise compared with children of healthy weight . Use of compensatory breathing strategies appeared to enable overweight children to avoid the experience of breathlessness at this intensity of exercise.

Suppression of adrenomedullin contributes to vascular leakage and altered epithelial repair during asthma.

BACKGROUND: The anti-inflammatory peptide, adrenomedullin (AM), and its cognate receptor are expressed in lung tissue, but its pathophysiological significance in airway inflammation is unknown. OBJECTIVES: This study investigated whether allergen-induced airway inflammation involves an impaired local AM response. METHODS: Airway AM expression was measured in acute and chronically sensitized mice following allergen inhalation and in airway epithelial cells of asthmatic and nonasthmatic patients. The effects of AM on experimental allergen-induced airway inflammation and of AM on lung epithelial repair in vitro were investigated. RESULTS: Adrenomedullin mRNA levels were significantly (P < 0.05) reduced in acute ovalbumin (OVA)-sensitized mice after OVA challenge, by over 60% at 24 h and for up to 6 days. Similarly, reduced AM expression was observed in two models of chronic allergen-induced inflammation, OVA- and house dust mite-sensitized mice. The reduced AM expression was restricted to airway epithelial and endothelial cells, while AM expression in alveolar macrophages was unaltered. Intranasal AM completely attenuated the OVA-induced airway hyperresponsiveness and mucosal plasma leakage but had no effect on inflammatory cells or cytokines. The effects of inhaled AM were reversed by pre-inhalation of the putative AM receptor antagonist, AM ((22-52)) . AM mRNA levels were significantly (P < 0.05) lower in human asthmatic airway epithelial samples than in nonasthmatic controls. In vitro, AM dose-dependently (10(-11) -10(-7) M) accelerated experimental wound healing in human and mouse lung epithelial cell monolayers and stimulated epithelial cell migration. CONCLUSION: Adrenomedullin suppression in T(H) 2-related inflammation is of pathophysiological significance and represents loss of a factor that maintains tissue integrity during inflammation.

Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.

When do infants and young children with cystic fibrosis acquire infection with Pseudomonas aeruginosa? Can this be eradicated when first detected? Children <6 yrs of age participated in an annual bronchoalveolar lavage (BAL)-based microbiological surveillance programme in Perth, Australia. When P. aeruginosa was detected, an eradication programme using combination treatment with i.v., oral and nebulised antibiotics was undertaken. Repeat BAL was performed 3 months following treatment, to assess eradication success. P. aeruginosa was detected in 33 (28.4%) children; median (range) age at detection was 30.5 (3.3-71.4) months. P. aeruginosa was mucoid at detection in six (18.2%) out of 33 patients and associated with respiratory symptoms in 16 (48.5%) out of 33 children. In total, 26 children underwent eradication therapy, with P. aeruginosa eradicated in 20 (77%) out of 26 following one eradication cycle and in three (total 88%) additional children following a second cycle. Eradication was associated with a significant decrease in neutrophil elastase and interleukin-1beta in BAL fluid 12 months post eradication. Eradication of Pseudomonas aeruginosa infection is achievable in young children with cystic fibrosis for up to 5 yrs using combination i.v., oral and nebulised antibiotic therapy and is associated with reduced pulmonary inflammation 12 months post eradication.

Forced oscillations in the clinical setting in young children with neonatal lung disease.

The extent of respiratory dysfunction is not well characterised in children with neonatal chronic lung disease (nCLD) too young to perform spirometry. Forced oscillations are easily performed by healthy young children; however, they may be more difficult for those with nCLD. The present study aimed to describe the feasibility of using the forced oscillation technique in children with nCLD in a routine clinical setting and to investigate the influence of neonatal factors on subsequent lung function. Respiratory function tests were attempted in 64 patients with nCLD aged 3.2-6.6 yrs. Respiratory resistance and reactance at 6, 8 and 10 Hz were expressed as z-scores derived from a healthy reference population. The within-test variation and between-test repeatability were also assessed. Technically, satisfactory data were obtained from 77% of children. On grouped data, z-scores for all oscillatory indices were different from zero and related to hospital oxygen administration in the neonatal period. In conclusion, the forced oscillation technique was feasible in preschool children with neonatal chronic lung disease in the clinical outpatient setting. These children had lung function significantly worse than that predicted from healthy children. Respiratory function assessed using forced oscillations appeared to reflect the severity of lung disease during the neonatal period.

Comparison of techniques for obtaining lower airway epithelial cells from children.

Airway epithelial cells (AECs) are important in asthma as they are the first cells to encounter pathogens/allergens. In children, AECs can be obtained using a "blind" nonbronchoscopic technique through an endotracheal tube. However, due to the increasing use of laryngeal masks the number of children in whom this technique is applicable has become limited. Recently, the present authors began to use a portable "bronchoscope-directed" technique to sample AECs. The current study compares both techniques in both asthmatic and nonasthmatic children. A total of 81 children undergoing elective surgery, were grouped according to atopic status and respiratory symptoms. Cellular yield of blind and bronchoscope-directed brushings were compared and immunocytochemistry performed. AECs were cultured and cytokine analysis of culture supernatant undertaken. Both techniques were equally well-tolerated, with the only adverse effect being a cough in 10% of the subjects. The mean+/-SD cell yield was higher in bronchoscope-directed than blind brushings (5.1+/-2.4 versus 3.1+/-1.4x10(6) cells). Immunocytochemistry confirmed an epithelial cell lineage. Culture supernatant cytokine concentrations were similar regardless of sampling technique with patterns preserved between asthmatic and healthy nonatopic phenotypes. Compared with blind brushing portable bronchoscope-directed brushing is well-tolerated, yields significantly more cells and is a potentially quick and useful technique for obtaining airway epithelial cells for research into childhood respiratory disease, specifically asthma.

Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells.

The stability of housekeeping genes (HKGs) is critical when performing real-time quantitative PCR. To date, the stability of common HKGs has not been systematically compared in human airway epithelial cells (AEC) in normal and atopic subjects. Expression levels of 12 HKGs were measured in AECs from a cohort of 30 healthy atopic nonasthmatic or atopic asthmatic children. Gene expression stability was determined using three different Visual Basic for Applications applets (geNorm, NormFinder and BestKeeper). All 12 HKGs were expressed in AECs. However, the hypoxanthine ribosyltransferase and TATA-binding protein genes were excluded from further analysis due to low expression levels. The cyclophilin A gene was ranked the most stable by all three methods. The expression levels of the beta-actin and glyceraldehyde-3-phosphate dehydrogenase genes were significantly different between the three groups of patients, with atopic asthmatics showing the highest expression levels for both genes. The results suggest that the cyclophilin A gene is the most suitable housekeeping gene analysed for expression studies utilising uncultured bronchial airway epithelial cells from healthy and asthmatic children, and highlight the importance of validating housekeeping genes for each experimental model.

Definition, assessment and treatment of wheezing disorders in preschool children: an evidence-based approach.

There is poor agreement on definitions of different phenotypes of preschool wheezing disorders. The present Task Force proposes to use the terms episodic (viral) wheeze to describe children who wheeze intermittently and are well between episodes, and multiple-trigger wheeze for children who wheeze both during and outside discrete episodes. Investigations are only needed when in doubt about the diagnosis. Based on the limited evidence available, inhaled short-acting beta(2)-agonists by metered-dose inhaler/spacer combination are recommended for symptomatic relief. Educating parents regarding causative factors and treatment is useful. Exposure to tobacco smoke should be avoided; allergen avoidance may be considered when sensitisation has been established. Maintenance treatment with inhaled corticosteroids is recommended for multiple-trigger wheeze; benefits are often small. Montelukast is recommended for the treatment of episodic (viral) wheeze and can be started when symptoms of a viral cold develop. Given the large overlap in phenotypes, and the fact that patients can move from one phenotype to another, inhaled corticosteroids and montelukast may be considered on a trial basis in almost any preschool child with recurrent wheeze, but should be discontinued if there is no clear clinical benefit. Large well-designed randomised controlled trials with clear descriptions of patients are needed to improve the present recommendations on the treatment of these common syndromes.

Dysregulated repair in asthmatic paediatric airway epithelial cells: the role of plasminogen activator inhibitor-1.

BACKGROUND: Asthma is associated with structural changes to airways such as extracellular matrix deposition and epithelial damage. Evidence suggests that asthmatic airway epithelial repair is abnormal and that elevated plasminogen activator inhibitor-1 levels observed in asthma may be involved in the epithelial repair process and in excessive matrix accumulation. OBJECTIVE: To assess the ability of asthmatic airway epithelial cells (AECs) to repair mechanically induced wounds and to investigate the role that plasminogen activator inhibitor-1 plays in the repair process. METHODS: AECs were isolated from atopic asthmatic and healthy non-atopic children by bronchial brushing, subcultured and wound repair experiments were performed. Plasminogen activator inhibitor-1 gene expression was assessed using real-time PCR while protein activity was measured in cell lysates as well as plasma. The role of plasminogen activator inhibitor-1 in epithelial proliferation and wound repair was investigated using siRNA. RESULTS: Cells from asthmatic children have a significantly longer repair time in comparison with cells from otherwise healthy donors. Plasminogen activator inhibitor-1 mRNA expression was up-regulated 68-fold in freshly isolated asthmatic cells compared with normal cells, and protein levels were also significantly elevated in the asthmatic cell lysates, but plasma levels were similar in both groups. Plasminogen activator inhibitor-1 cells expression increased in both cohorts during culture. Gene silencing substantially reduced the rate of proliferation in asthmatic and healthy cells. Mechanical wounding of epithelial monolayers induced plasminogen activator inhibitor-1 expression in asthmatic and non-asthmatic cohorts, while gene silencing delayed wound repair of healthy cell, with minimal effect on those from asthmatics. CONCLUSION: Asthmatic AECs are inherently dysfunctional in their ability to repair wounds; plasminogen activator inhibitor-1 mRNA and protein activity are constitutively up-regulated in asthmatic epithelium and play functional roles in both proliferation and repair of healthy cells. In asthmatic cells, elevated plasminogen activator inhibitors-1 levels fail to stimulate epithelial repair.

How free of tobacco smoke are 'smoke-free' homes?

UNLABELLED: The risks of exposure to environmental tobacco smoke (ETS) are well established and 'harm reduction' strategies such as smoking outside to protect infants and children from exposure to ETS have been advocated for some time. The aim of this study was to assess the validity of self-reported smoking levels in residential settings. The participants were families (n = 92) randomly selected from lower socioeconomic areas of Perth, Western Australia. Each household was monitored for vapor phase nicotine and particulates with an aerodynamic diameter of < or = 10 microm (PM(10)). Of the 42% (39) households who reported that someone smoked cigarettes at home, only four (4%) said that smoking occurred inside the house. There was a 'moderate' agreement between parental-reported tobacco smoking and levels of nicotine (kappa = 0.55, P < 0.01). There were significant differences in the median levels of air nicotine (P < 0.01) and PM(10) (P < 0.05) between households in which smoking was reported as only occurring outside, and the smoke-free households. PRACTICAL IMPLICATIONS: The study outcome suggests that a strategy based on the separation of children and smoking activity is inadequate to protect the former from ETS at home, and that health professionals should give parents unambiguous advice to give up smoking in order to make their homes a completely smoke-free environment.

CD14 C-159T and early infection with Pseudomonas aeruginosa in children with cystic fibrosis.

Early acquisition of Pseudomonas aeruginosa is associated with a poorer prognosis in patients with cystic fibrosis. We investigated whether polymorphisms in CD14, the lipopolysaccharide receptor, increase the risk of early infection. Forty-five children with cystic fibrosis were investigated with annual bronchoalveolar lavage (BAL) and plasma sCD14 levels. Plasma sCD14 levels were significantly lower in children from whom P.aeruginosa was subsequently isolated (492.75 microg/ml vs. 1339.43 microg/ml, p = 0.018). Those with the CD14 -159CC genotype had a significantly increased risk of early infection with P.aeruginosa suggesting that CD14 C-159T plays a role in determining the risk of early infection with P.aeruginosa.

Transmission of pressure across the chest wall during the rapid thoracic compression technique in infants.

During the rapid thoracic compression maneuver in infants, the transmission of pressure from compression jacket to pleural space and airway is less at functional residual capacity than at end inspiration. To examine whether reduced pressure transmission at functional residual capacity vs. higher lung volumes is explained by passive characteristics of the chest wall rather than by respiratory muscle activity, we assessed the pressure transmitted across the chest wall in nine anesthetized infants and young children after muscle relaxation. We measured esophageal and airway occlusion pressure during chest compressions at different lung volumes determined by varying distending pressure. In six subjects studied under static conditions, there was an approximately linear relationship between distending pressure and the proportion of pressure transmitted to the airway and esophagus from the compression jacket. The mean r2 value (95% confidence interval) was 0.80 (0.09) for pressure transmission to the airway and 0.85 (0.04) for pressure transmission to the esophagus. This relationship between lung volume and pressure transmission observed under static conditions was also demonstrated dynamically. Thus the reduced transmission of pressure from compression jacket to airway and pleural space at low lung volumes occurs independently of respiratory muscle activity.

Is breathing in infants chaotic? Dimension estimates for respiratory patterns during quiet sleep.

We describe an analysis of dynamic behavior apparent in times-series recordings of infant breathing during sleep. Three principal techniques were used: estimation of correlation dimension, surrogate data analysis, and reduced linear (autoregressive) modeling (RARM). Correlation dimension can be used to quantify the complexity of time series and has been applied to a variety of physiological and biological measurements. However, the methods most commonly used to estimate correlation dimension suffer from some technical problems that can produce misleading results if not correctly applied. We used a new technique of estimating correlation dimension that has fewer problems. We tested the significance of dimension estimates by comparing estimates with artificial data sets (surrogate data). On the basis of the analysis, we conclude that the dynamics of infant breathing during quiet sleep can best be described as a nonlinear dynamic system with large-scale, low-dimensional and small-scale, high-dimensional behavior; more specifically, a noise-driven nonlinear system with a two-dimensional periodic orbit. Using our RARM technique, we identified the second period as cyclic amplitude modulation of the same period as periodic breathing. We conclude that our data are consistent with respiration being chaotic.

Validation of respiratory inductance plethysmography ("Respitrace") for the measurement of tidal breathing parameters in newborns.

INTRODUCTION: The ratio of the time to reach peak (maximum) tidal expiratory flow (Tme) to total expiratory time (Te) is smaller in infants who later develop lower respiratory tract disease. In previous studies infants have been sedated and flow measured using a pneumotachograph with face-mask. These methodological factors are known to affect tidal breathing, and the frequent need for sedation limits the use of the technique to relatively small studies. The aim of this study was to validate uncalibrated respiratory inductance plethysmography (Respitrace) to measure Tme/Te in unsedated newborns. METHODS: Nineteen normal term infants were studied during quiet sleep. Agreement between Tme/Te measured directly using a pneumotachograph and with Respitrace was assessed in 15 infants. Repeatability of the Respitrace technique was assessed in 10 infants. RESULTS: The mean Tme/Te for the 19 infants using Respitrace was 0.46 (S.D. 0.14). The mean difference between Tme/Te obtained using Respitrace and that measured with a pneumotachograph was 0.014; 95% of Respitrace readings were between -0.042 and 0.070 of the pneumotachograph values. The mean difference between repeat Respitrace values was 0.02 with 95% of the second measurements within 0.066 of the first. CONCLUSIONS: These results indicate that Respitrace can be used to determine Tme/Te accurately.

Lung function and bronchial challenges in infants: repeatability of histamine and comparison with methacholine challenges.

UNLABELLED: Limited information exists regarding the repeatability of lung function and bronchial challenge tests using the rapid thoracic compression technique (RTC) in infants. AIMS: To determine the repeatability of lung function and histamine challenge test results using the RTC technique and to compare the results obtained for bronchial challenges using histamine (H) and methacholine (M). METHODS: Twelve infants [7 healthy, 5 with cystic fibrosis (CF) had pairs of H challenges 1 week apart. Eleven infants (7 healthy, 4 CF) had one H and one M challenge a week apart. The provocative concentration of H or M to cause a 40% fall in maximum flow at functional residual capacity (PC40) was determined using the RTC technique. Twenty-three comparisons were possible between maximal expiratory flow at functional residual capacity (VmaxFRC) measurements made 1 week apart. RESULTS: The mean difference between pairs of VmaxFRC measurements was 6.4% of baseline, and the coefficient of repeatability was 31.1% of baseline. The mean difference between PC40(H) measurements was 0.163 doubling concentrations, with a coefficient of repeatability of 1.66 doubling concentrations. The mean difference between PC40(H) and PC40(M) was 0.75 doubling concentrations, with 95% of PC40(H) between -0.18 to 1.69 doubling concentrations of the PC40(M). CONCLUSIONS: Although the repeatability of VmaxFRC using the RTC technique is less than for voluntary forced expiratory flow parameters in older children, similar results were obtained for infants as observed in older subjects for repeatability of H challenges and agreement between measures of bronchial responsiveness using H or M.

The effect of age on oxygen desaturation during histamine inhalation challenge in normal infants.

Arterial oxygen saturation (SaO2) is usually measured during inhalation challenges in infants as desaturation has been demonstrated with provoked bronchoconstriction. We wished to examine whether measurement of SaO2 would provide a simple noninvasive indicator of respiratory function (RF) changes occurring during inhalation challenge in infants. Histamine inhalation challenges were performed longitudinally in 22 normal healthy infants at 4 and 26 weeks of age. RF was measured by calculating maximum forced expiratory flow at functional residual capacity (VmaxFRC) using the rapid thoracic compression technique. Airway responsiveness was assessed using histamine; the provoking concentration (PC) was that which caused a fall in VmaxFRC of at least 40% from baseline. The provocative concentration for an exact fall of 40% in VmaxFRC (PC40) from baseline was derived by linear interpolation. SaO2 was continuously monitored by pulse oximetry. One month old infants had higher SaO2 levels throughout the inhalation challenge in comparison to their SaO2 levels at 6 months of age. Significant falls in SaO2 were observed at the PC at both ages. However, at the age of 26 weeks the infants had greater median falls in SaO2 [PC, 4.5% (95%CI: 3.0, 7.0)] compared to the response at age 4 weeks [3.0% (95%CI: 1.0, 4.0) (P < 0.01)]. Median falls in VmaxFRC at the PC were not different between the ages. These results indicate an age-dependent discordance between airway and SaO2 response in healthy infants during histamine-induced bronchoconstriction.

Perceptual feature sorting of brain-injured patients: left versus right hemisphere preferences.

A 20-item Piagetian-type sorting task was presented to 12 medically verified unilaterally brain-injured patients, whose performances were compared to 12 non-brain-injured controls. Cerebrovascular involvement accounted for the brain injuries. Speech and language status was determined by use of the short form of the Minnesota Test for Differential Diagnosis of Aphasia (Schuell, 1965). Five subjects had right hemisphere involvement. Of the seven subjects with left hemisphere involvement, four were fluent, two were nonfluent, and one had no appreciable speech or language difficulties. The left hemisphere brain-injured subjects and the controls showed preferences for the features of shape and texture. In contrast, the right hemisphere brain-injured subjects preferred the feature of size.