iNKT cells are increased in children with severe therapy-resistant asthma.

BACKGROUND: Invariant natural killer T (iNKT) cells play complex functions in the immune system, releasing both Th1 and Th2 cytokines. The role of iNKT cells in human asthma is still controversial and never described in severe therapy-resistant asthma in children. The objective of this work was to analyse iNKT frequency in peripheral blood of children with severe therapy-resistant asthma (STRA), compared to children with milder asthma and healthy controls. METHODS: Children with asthma (n=136) (non-severe and STRA) from a referral centre and healthy controls (n=40) were recruited. Peripheral blood mononuclear cells were isolated, stained with anti-CD3 and anti-iNKT (Vα24Jα18), and analysed through flow cytometry. Atopic status was defined by measuring specific IgE in serum. Airway inflammation was assessed by induced sputum. RESULTS: Children with asthma presented an increased frequency of CD3+iNKT+ cells (median 0.38% IQR 0.18-1.9), compared to healthy controls (median 0.26% IQR 0.10-0.43) (p=0.025). Children with STRA also showed an increased frequency of iNKT cells (1.5% IQR 1.05-2.73) compared to healthy controls and non-severe asthmatic children (0.35% IQR 0.15-1.6; p=0.002). The frequency of iNKT cells was not different between atopic and non-atopic children. In addition, iNKT cells were not associated with any inflammatory pattern of induced sputum studied. CONCLUSION: Our data suggests that iNKT cells play a role in paediatric asthma, which is also associated with the severity of disease, but independent of the atopic status.

Input respiratory impedance in mice: comparison between the flow-based and the wavetube method to perform the forced oscillation technique.

Objective and approach: In this study, we estimated the constant phase model (CPM) parameters from the respiratory impedance of male BALB/c mice by performing the forced oscillation technique (FOT) in a control group (n = 8) and in a murine model of asthma (OVA) (n = 10). Then, we compared the results obtained by two different methods, using a commercial equipment (flexiVent-flexiWare 7.X; SCIREQ, Montreal, Canada) (FXV) and a wavetube method equipment (Sly et al 2003 J. Appl. Physiol. 94 1460-6) (WVT). We believe that the results from different methods may not be comparable. First, we compared the results performing a two-way analysis of variance (ANOVA) for the resistance, elastance and tissue damping. MAIN RESULTS: We found statistically significant differences in all CPM parameters, except for resistance, when comparing Control and OVA groups. When comparing devices, we found statistically significant differences in resistance, while differences in elastance were not observed. For tissue damping, the results from WVT were observed to be higher than those from FXV. Finally, when comparing the relative variation between the CPM parameters of the Control and OVA groups in both devices, no significant differences were observed for all parameters. SIGNIFICANCE: We then conclude that this assessment can compensate the effect of using different cannulas. Furthermore, tissue damping differences between groups can be compensated, since bronchoconstrictors were not used. Therefore, we believe that relative variations in the results between groups can be a comparing parameter when using different equipment without bronchoconstrictor administration.

Immune response of toddlers with history of prematurity.

BACKGROUND: It is not quite well established how immune responses differ in term and preterm infants beyond the first year of life. This study aimed to evaluate aspects of the innate and adaptive immune responses in a group of preterm infants in comparison with their term peers. METHODS: In this cross-sectional study peripheral blood mononuclear cells (PBMC) were isolated from preterm and term children at age three years. Innate immune response was evaluated by the analysis of TLR receptors expression on CD11c+HLADRhigh cells and inflammatory cytokine production after PBMC stimulation with Toll like receptors (TLR) ligands. Adaptive immune response was evaluated by T cells' phenotyping and function after stimulation with polyclonal conventional T cell stimulus. CONCLUSION: We have found that the patterns of innate and adaptive immune responses at 3 years of age were not affected by the fact of the children having being born preterm or at term.

Bacterial extract (OM-85) with human-equivalent doses does not inhibit the development of asthma in a murine model.

BACKGROUND: OM-85 is an immunostimulant bacterial lysate, which has been proven effective in reducing the number of lower airways infections. We investigated the efficacy of the bacterial lysate OM-85 in the primary prevention of a murine model of asthma. METHODS: In the first phase of our study the animals received doses of 0.5µg, 5µg and 50µg of OM-85 through gavage for five days (days -10 to -6 of the protocol), 10 days prior to starting the sensitisation with ovalbumin (OVA), in order to evaluate the results of dose-response protocols. A single dose (5µg) was then chosen in order to verify in detail the effect of OM-85 on the pulmonary allergic response. Total/differential cells count and cytokine levels (IL-4, IL-5, IL-13 and IFN-γ) from bronchoalveolar lavage fluid (BALF), OVA-specific IgE levels from serum, lung function and lung histopathological analysis were evaluated. RESULTS: OM-85 did not reduce pulmonary eosinophilic response, regardless of the dose used. In the phase protocol using 5µg/animal of OM-85, no difference was shown among the groups studied, including total cell and eosinophil counts in BALF, serum OVA-specific IgE, lung histopathologic findings and lung resistance. However, OM-85 decreased IL-5 and IL-13 levels in BALF. CONCLUSIONS: OM-85, administered in early life in mice in human-equivalent doses, does not inhibit the development of allergic pulmonary response in mice.

Respiratory syncytial virus induces phosphorylation of mTOR at ser2448 in CD8 T cells from nasal washes of infected infants.

Respiratory syncytial virus (RSV)-specific CD8(+) T cell responses do not protect against reinfection. Activation of mammalian target of rapamycin (mTOR) impairs memory CD8(+) T cell differentiation. Our hypothesis was that RSV inhibits the formation of CD8(+) T cells memory responses through mTOR activation. To explore this, human and mouse T cells were used. RSV induced mTOR phosphorylation at Ser2448 in CD8 T cells. mTOR activation by RSV was completely inhibited using rapamycin. RSV-infected children presented higher mTOR gene expression on nasal washes comparing to children infected with metapneumovirus and rhinovirus. In addition, RSV-infected infants presented a higher frequency of CD8(+) pmTORser2448(+) T cells in nasal washes compared to RSV-negative infants. Rapamycin treatment increased the frequency of mouse CD8 RSV-M282-90 pentamer-positive T cells and the frequency of RSV-specific memory T cells precursors. These data demonstrate that RSV is activating mTOR directly in CD8 T cells, indicating a role for mTOR during the course of RSV infection.

Effect of different helminth extracts on the development of asthma in mice: The influence of early-life exposure and the role of IL-10 response.

It is not currently clear whether different parasites have distinct effects on the airway inflammatory response in asthma and whether exposure in early life to helminths have a stronger impact in a potential inhibitory effect on asthma. The aim of this study is to evaluate the effect of exposure to different helminth extracts on the development of allergic pulmonary response in mice, including early-life exposure. Different helminth extracts (Angiostrongylus costaricensis, Angiostrongylus cantonensis and Ascaris lumbricoides) were studied in female adult BALB/c and C57BL/6 IL-10-deficient mice in a protocol of murine asthma, injected intraperitoneally in different periods of exposure (early, pre-sensitization and post-sensitization). Cell counts in bronchoalveolar lavage (BAL), eosinophil peroxidase (EPO) from lung tissue, cytokine levels from BAL/spleen cell cultures, and lung histology were analyzed. Airway cellular influx induced by OVA was significantly inhibited by extracts of A. cantonensis and A. lumbricoides. Extracts of A. lumbricoides and A. costaricensis led to a significant reduction of IL-5 in BAL (p < 0.001). Only the exposure to A. lumbricoides led to an increased production of IL-10 in the lungs (p < 0.001). In IL-10-deficient mice exposed to A. costaricensis pre-sensitization, eosinophil counts and IL-5 levels in BAL and EPO in lung tissue were significantly reduced. In the early exposure to A. cantonensis, lung inflammation was clearly inhibited. In conclusion, different helminth extracts inhibit allergic lung inflammation in mice. IL-10 may not play a central role in some helminth-host interactions. Early exposure to helminth extracts could be a potential strategy to explore primary prevention in asthma.

Extracellular DNA traps in bronchoalveolar fluid from a murine eosinophilic pulmonary response.

Asthma is associated with a loss of the structural integrity of airway epithelium and dysfunction of the physical barrier, which protects airways from external harmful factors. Granulocyte activation causes the formation of extracellular traps, releasing web-like structures of DNA and proteins, being important to kill pathogens extracellularly. We investigated whether eosinophils infiltrating airways in an experimental model of asthma would induce eosinophil extracellular traps (EETs) in bronchoalveolar lavage fluid and lung tissue. We showed that an ovalbumin (OVA) asthma protocol presented a significant increase in eosinophil counts with increased extracellular DNA in bronchoalveolar lavage fluid as well as in lung tissue, confirming the presence of DNA traps colocalized with eosinophil peroxidase. EETs formation was reversed by DNase treatment. With these approaches, we demonstrated for the first time that OVA-challenged mice release extracellular DNA traps, which could aggravate pulmonary dysfunction.

Plasma brain-derived neurotrophic factor levels are associated with clinical severity in school age children with asthma.

BACKGROUND: Asthma is characterized by chronic inflammation of the airways with significant changes in leucocyte trafficking, cellular activation and tissue remodelling. Brain-derived neurotrophic factor (BDNF) has been involved with asthma and allergic diseases but its role as a severity marker in paediatric asthma has not been clinically assessed. OBJECTIVES: To evaluate plasma BDNF and inflammatory markers in order to address their relationships with disease severity in children (6-15 years) with controlled persistent asthma. METHODS: Children with persistent asthma were selected and lung function and skin prick tests were performed in all patients. Plasma BDNF levels and various inflammatory markers (CCL3, CCL11, CCL22, CCL24, CXCL8, CXCL9, CXCL10, soluble TNF receptors) were assessed by ELISAs. RESULTS: Subjects with moderate and severe asthma had higher BDNF levels than mild asthma and controls (P<0.001). The chemokines studied and soluble TNF receptors did not differ between the studied groups. CONCLUSION AND CLINICAL RELEVANCE: Our results indicate BDNF as a potential biomarker for clinical severity in children with asthma.

Neutrophilic airway inflammation is a main feature of induced sputum in nonatopic asthmatic children.

BACKGROUND: Asthma phenotypes are well described among children. However, there are few studies comparing airway inflammation in different clinical presentations of pediatric asthma. We tested the hypothesis that nonatopic asthma is associated with a predominant noneosinophilic inflammation in the airways, as assessed by induced sputum. The objective of this study was to evaluate the cytological characteristics of induced sputum (IS) in atopic (AA), nonatopic asthmatics (NAA) and nonatopic nonasthmatic children (NANA). METHODS: Of 90 selected children, 77 met eligibility criteria for performing IS and were classified as: AA, n = 28, NAA, n = 29 and NANA, n = 19. Subjects answered to a set of ISAAC-based questions and were skin-tested for common aeroallergens. A defined series of exclusion criteria was applied. RESULTS: Induced sputum was obtained from 54 (70.1%) subjects (21 AA, 20 NAA and 13 NANA). Demographic data and mean FEV(1) were similar in the three groups. The proportion of eosinophils [median, inter quartile range (IQR)] was significantly higher in the sputum of AA [(6.0.)12)] compared with NAAs [0 (2)] and NANAs [0 (1)], P < 0.001. The proportion of children with sputum eosinophilia (eos > 3%) was also significantly higher in AA (71.4%) when compared with NAA (28.6%); none of the NANA had sputum eosinophilia. Nonatopic asthmatic children had significantly higher proportions and absolute number of neutrophils than AA and controls. CONCLUSIONS: The results suggest that nonatopic children present IS with a cell pattern that is predominantly neutrophilic while eosinophilia is the hallmark of airway inflammation in the majority of atopic wheezing children not treated with inhaled steroids.

TNF-alpha and IL-10 levels in tracheobronchial lavage of ventilated preterm infants and subsequent lung function.

The role of airway inflammation in ventilated preterm newborns and the risk factors associated with the development of chronic lung disease are not well understood. Our objective was to analyze the association of the airway inflammatory response in ventilated preterm infants by serial measurements of TNF-alpha and IL-10 in tracheobronchial lavage (TBL) with perinatal factors and lung function measured early in life. A series of TBL samples were collected from ventilated preterm infants (less than 32 weeks of gestational age) and concentrations of TNF-alpha and IL-10 were measured by ELISA. Pulmonary function tests were performed after discharge by the raised volume rapid compression technique. Twenty-five subjects were recruited and 70 TBL samples were obtained. There was a significant positive association between TNF-alpha and IL-10 levels and length of time between the rupture of the amniotic membranes and delivery (r = 0.65, P = 0.002, and r = 0.57, P < 0.001, respectively). Lung function was measured between 1 and 22 weeks of corrected age in 10 patients. Multivariable analysis with adjustment for differences in lung volume showed a significant negative association between TNF-alpha levels and forced expiratory flow (FEF(50); r = -0.6; P = 0.04), FEF(75) (r = -0.76; P = 0.02), FEF(85) (r = -0.75; P = 0.03), FEF(25-75) (-0.71; P = 0.02), and FEV(0.5) (r = -0.39; P = 0.03). These data suggest that TNF-alpha levels in the airways during the first days of life were associated with subsequent lung function abnormalities measured weeks or months later.

TNF-a and IL-10 levels in tracheobronchial lavage of ventilated preterm infants and subsequent lung function

The role of airway inflammation in ventilated preterm newborns and the risk factors associated with the development of chronic lung disease are not well understood. Our objective was to analyze the association of the airway inflammatory response in ventilated preterm infants by serial measurements of TNF-a and IL-10 in tracheobronchial lavage (TBL) with perinatal factors and lung function measured early in life. A series of TBL samples were collected from ventilated preterm infants (less than 32 weeks of gestational age) and concentrations of TNF-a and IL-10 were measured by ELISA. Pulmonary function tests were performed after discharge by the raised volume rapid compression technique. Twenty-five subjects were recruited and 70 TBL samples were obtained. There was a significant positive association between TNF-a and IL-10 levels and length of time between the rupture of the amniotic membranes and delivery (r = 0.65, P = 0.002, and r = 0.57, P < 0.001, respectively). Lung function was measured between 1 and 22 weeks of corrected age in 10 patients. Multivariable analysis with adjustment for differences in lung volume showed a significant negative association between TNF-a levels and forced expiratory flow (FEF50; r = -0.6; P = 0.04), FEF75 (r = -0.76; P = 0.02), FEF85 (r = -0.75; P = 0.03), FEF25-75 (-0.71; P = 0.02), and FEV0.5 (r = -0.39; P = 0.03). These data suggest that TNF-a levels in the airways during the first days of life were associated with subsequent lung function abnormalities measured weeks or months later.

Nonatopic asthma is associated with helminth infections and bronchiolitis in poor children.

Asthma is common in urban centres in Latin America, but atopic asthma may not be the main phenotype among children. Helminth infections are highly prevalent in poor populations, and it was hypothesised that they attenuate allergic asthma, whereas other factors are related to the expression of a nonatopic wheeze/asthma phenotype. A total of 1,982 children from Southern Brazil with a mean+/-sd age of 10.1+/-0.76 yrs completed asthma questionnaires, and 1,011 were evaluated for intestinal parasites and atopy using skin-prick tests (SPTs). Wheeze in the previous 12 months was reported by 25.6%, and 9.3% showed current asthma; 13% were SPT-positive and 19.1% were positive for any helminths. Most children with either wheeze or asthma were SPT-negative; however, severe wheeze was more prevalent among the atopic minority. Helminth infections were inversely associated with positive SPT results. Bronchiolitis before the age of 2 yrs was the major independent risk factor for asthma at age 10 yrs; high-load Ascaris infection, a family history of asthma and positive SPT results were also asthma risk factors. Most asthma and wheeze are of the nonatopic phenotype, suggesting that some helminths may exert an attenuating effect on the expression of the atopic portion of the disease, whereas viral bronchiolitis predisposes more specifically to recurrent airway symptoms.

Nasal wash as an alternative to bronchoalveolar lavage in detecting early pulmonary inflammation in children with cystic fibrosis.

OBJECTIVE: The aim of this study was to determine whether nasal inflammation reflects pulmonary inflammation in young children with cystic fibrosis (CF), as assessed by inflammatory markers in nasal wash (NW) and bronchoalveolar lavage (BAL), respectively. METHODS: CF patients younger than 6 years of age who were to undergo bronchoscopy for routine BAL from May 2000 to October 2001 were recruited for this study. NW was collected immediately after the patient was sedated for bronchoscopy. Total cell counts (TCC), differential cell counts and interleukin (IL)-8 levels (enzyme-linked immunosorbent assay) were assessed in NW and BAL. RESULTS: In total, 19 children with CF (mean age, 1.9 years; SD, 1.7 years) were included in the study. There was a significant relationship between IL-8 and the percentages of neutrophils in NW (r (2) = 0.76; P < 0.001) and in BAL fluid (r 2 = 0.62; P = 0.006). Similarly, IL-8 concentrations in the NW correlated with those in the BAL (r 2 = 0.48; P = 0.036) and neutrophil percentages in NW correlated significantly with those in BAL (r 2 = 0.7; P = 0.004). CONCLUSION: When measured under 'ideal' conditions, nasal IL-8 reflects lower airway levels and may reflect the inflammatory stimulus that results in neutrophilic inflammation. These data encourage further assessment of nasal wash under clinically appropriate conditions to determine its utility for assessing inflammation in young children with CF.

Correlation of forced oscillation technique in preschool children with cystic fibrosis with pulmonary inflammation.

BACKGROUND: Lung disease in cystic fibrosis (CF) is established in early childhood with recurrent bacterial infections and inflammation. Using spirometry, the effect of this early lung damage cannot be measured until a child is 6 years of age when some irreversible lung damage may already have occurred. Techniques for measurement of lung function in infants and young children include raised volume rapid thoracic compression (RVRTC) and low frequency forced oscillation (LFFOT). The aim of this study was to investigate the role of inflammation and infection on a population of infants and young children with CF and to determine whether lung function in this population (measured by LFFOT) is affected by early lung disease. METHODS: Lung function was measured by LFFOT in 24 children undergoing bronchoalveolar lavage (BAL) on 27 occasions as part of an annual programme while still under general anaesthesia. Following lung function testing, three aliquots of saline were instilled into the right middle or lower lobe. The first aliquot retrieved was processed for the detection of microbes, and the remaining aliquots were pooled to assess inflammatory markers (cytology, IL-8, NE, LTB(4)). RESULTS: Inflammation (percentage and number of neutrophils) was significantly higher in children with infections (p<0.001, p = 0.04, respectively), but not in those with symptoms. Several markers of inflammation significantly correlated with LFFOT parameters (R, G, and eta). CONCLUSION: Infections and inflammation are established before symptoms are apparent. Inflammation is correlated with measures of parenchymal changes in lung function measured by LFFOT.

Th-1 and Th-2 cytokine production in infants with virus-associated wheezing.

Wheezing associated with respiratory viral infections in infancy is very common and results in high morbidity worldwide. The Th1/Th2 pattern of immune response in these patients remains unclear and previous studies have shown controversial results. The aim of the present study was to compare the type of Th1/Th2 cytokine response between infants with acute bronchiolitis, recurrent wheezing and upper respiratory infections from a developing country. Infants younger than 2 years of age admitted to Hospital São Lucas, Porto Alegre, RS, Brazil, between May and November 2001, with an acute episode of wheezing associated with viral respiratory infection were selected. Subjects with upper respiratory infections from the emergency department were selected for the control group. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels from nasal aspirates were determined by ELISA from peripheral mononuclear cell cultures. Twenty-nine subjects with acute bronchiolitis, 18 with recurrent wheezing and 15 with upper respiratory infections were enrolled. There were no differences in family history of atopy or parental smoking between groups. Oxygen requirement was similar for the acute bronchiolitis and recurrent wheezing groups. The percentage of positive tests for the cytokines studied and the IFN-gamma/IL-4 ratio was similar for all groups. Comparison of the polarized Th1/Th2 cytokine results for the various groups showed no specific pattern of cytokine production. Infants with wheezing from a developing country do not show any specific predominant pattern of Th1/Th2 cytokine production, suggesting that multiple factors may be involved in the pathogenesis of this illness.

Th-1 and Th-2 cytokine production in infants with virus-associated wheezing

Wheezing associated with respiratory viral infections in infancy is very common and results in high morbidity worldwide. The Th1/Th2 pattern of immune response in these patients remains unclear and previous studies have shown controversial results. The aim of the present study was to compare the type of Th1/Th2 cytokine response between infants with acute bronchiolitis, recurrent wheezing and upper respiratory infections from a developing country. Infants younger than 2 years of age admitted to Hospital São Lucas, Porto Alegre, RS, Brazil, between May and November 2001, with an acute episode of wheezing associated with viral respiratory infection were selected. Subjects with upper respiratory infections from the emergency department were selected for the control group. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels from nasal aspirates were determined by ELISA from peripheral mononuclear cell cultures. Twenty-nine subjects with acute bronchiolitis, 18 with recurrent wheezing and 15 with upper respiratory infections were enrolled. There were no differences in family history of atopy or parental smoking between groups. Oxygen requirement was similar for the acute bronchiolitis and recurrent wheezing groups. The percentage of positive tests for the cytokines studied and the IFN-gamma/IL-4 ratio was similar for all groups. Comparison of the polarized Th1/Th2 cytokine results for the various groups showed no specific pattern of cytokine production. Infants with wheezing from a developing country do not show any specific predominant pattern of Th1/Th2 cytokine production, suggesting that multiple factors may be involved in the pathogenesis of this illness.

Infection of BALB/c mice with Angiostrongylus costaricensis decreases pulmonary inflammatory response to ovalbumin.

SUMMARY The prevalence of asthma in developing countries is lower than in developed countries. Viral, bacterial and parasitic infections may be associated with this discrepancy. The relationship between parasitic infection and asthma prevalence is not clear. Previous controversial data have demonstrated that parasitic infection may either predispose or protect against the development of asthma. The aim of this study is to determine whether infection with Angiostrongylus costaricensis (A. costaricensis) decreases inflammatory lung response to ovalbumin (OVA) in mice. Seven BALB/c mice were infected with A. costaricensis by orogastric gavage (10 larvae/mouse) on day (D) 0. The mice were immunized against OVA by intraperitoneal injection on D 5 and D 12 and received an intranasal OVA challenge (40 micro L) on D 15 and D 17. On D 19 bronchoalveolar lavage (BAL) was performed. Six BALB/c mice (control group) were immunized with OVA using the same protocol, but were not infected with A. costaricensis. Interleukin (IL)-1beta and IL-6 levels were measured in the BAL fluid by using commercial ELISA assays. Total cell counts and differential cell counts were performed in the BAL fluid samples. The group infected with A. costaricensis had lower total cell count in the BAL fluid when compared with the control group (0.11 x 10(6)cells/mL and 0.3 x 10(6)cells/mL, respectively; P = 0.013). BAL fluid IL-1beta levels in the infected group were significantly lower than in the control group (P = 0.008). IL-6 levels in BAL fluid were not different between the groups studied. We conclude that Angiostrongylus costaricensis infection in mice decreases pulmonary inflammatory response to OVA.

[Severe acute asthma in the child] / Asma aguda grave na criança.

OBJECTIVE: To present a current review about diagnosis and treatment of severe acute asthma, based on the reviewed publications and the authors personal experience. METHODS: We reviewed the most relevant articles about diagnosis and management of severe acute asthma. RESULTS: Severe acute asthma is an important cause of morbidity in children and it can usually lead to respiratory failure. The diagnostic (clinical manifestations and lung function tests) and treatment (oxygen, bronchodilators and steroids) are now well determined by the literature, and are very important for a good prognosis of every patient. CONCLUSIONS: Considering severe acute asthma a prevalent condition in pediatric emergency room and intensive care unit, the ability to make an early diagnosis of this condition, associated with a prompt treatment, are essential for an effective reduction in morbimortality rates.