Atul Gupta

Relationship between Serum Vitamin D, Disease Severity, and Airway Remodeling in Children with Asthma

RATIONALE Little is known about vitamin D status and its effect on asthma pathophysiology in children with severe, therapy-resistant asthma (STRA). OBJECTIVES Relationships between serum vitamin D, lung function, and pathology were investigated in pediatric STRA. METHODS Serum 25-hydroxyvitamin D [25(OH)D(3)] was measured in 86 children (mean age, 11.7 yr): 36 with STRA, 26 with moderate asthma (MA), and 24 without asthma (control subjects). Relationships between 25(OH)D(3), the asthma control test (ACT), spirometry, corticosteroid use, and exacerbations were assessed. Twenty-two of 36 children with STRA underwent fiberoptic bronchoscopy, bronchoalveolar lavage, and endobronchial biopsy with assessment of airway inflammation and remodeling. MEASUREMENTS AND MAIN RESULTS 25(OH)D(3) levels (median [IQR]) were significantly lower in STRA (28 [22-38] nmol/L) than in MA (42.5 [29-63] nmol/L) and control subjects (56.5 [45-67] nmol/L) (P < 0.001). There was a positive relationship between 25(OH)D(3) levels and percent predicted FEV(1) (r = 0.4, P < 0.001) and FVC (r = 0.3, P = 0.002) in all subjects. 25(OH)D(3) levels were positively associated with ACT (r = 0.6, P < 0.001), and inversely associated with exacerbations (r = -0.6, P < 0.001) and inhaled steroid dose (r = -0.39, P = 0.001) in MA and STRA. Airway smooth muscle (ASM) mass, but not epithelial shedding or reticular basement membrane thickness, was inversely related to 25(OH)D(3) levels (r = -0.6, P = 0.008). There was a positive correlation between ASM mass and bronchodilator reversibility (r = 0.6, P = 0.009) and an inverse correlation between ASM mass and ACT (r = -0.7, P < 0.001). CONCLUSIONS Lower vitamin D levels in children with STRA were associated with increased ASM mass and worse asthma control and lung function. The link between vitamin D, airway structure, and function suggests vitamin D supplementation may be useful in pediatric STRA.

Pediatric severe asthma is characterized by eosinophilia and remodeling without TH2 cytokines

BACKGROUND The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood. OBJECTIVES We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the T(H)2 cytokines IL-4, IL-5, and IL-13. METHODS Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen T(H)2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation. RESULTS Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5(+) and IL-13(+) cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid T(H)2 cytokines had significantly lower lung function than those with undetectable BAL fluid T(H)2 cytokines. CONCLUSIONS STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the T(H)2 mediators that are thought to drive allergic asthma were mostly absent.

The role of 1α,25-dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+and IL-10+ CD4+ T cells

1α,25‐Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL‐10‐secreting CD4+ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3+ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3+ and IL‐10+ CD4+T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL‐10 at more moderate levels, with little coexpression of these molecules. The Foxp3+ and IL‐10+ T‐cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL‐10. 1α25VitD3 enables the selective expansion of Foxp3+ Treg cells over their Foxp3− T‐cell counterparts. Equally, 1α25VitD3 maintains Foxp3+ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4+Foxp3+ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL‐10+ and Foxp3+ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.

Vitamin D and Asthma in Children

Vitamin D deficiency and insufficiency are increasingly being recognized in the general population, and have been largely attributed to lifestyle changes (reduced exposure to sunshine due to working indoors or the use of protective clothing and sunscreen; changes in diet) over the last few decades. The musculoskeletal consequences of severe vitamin D deficiency are well established, however, a number of other disorders have now been linked to vitamin D insufficiency, including asthma. There is growing appreciation of the likely importance of vitamin D as a pleiotrophic mediator that contributes to pulmonary health. Children with asthma appear to be at increased risk of vitamin D insufficiency. Epidemiologic data suggest that low serum vitamin D in children with asthma is associated with more symptoms, exacerbations, reduced lung function, increased medication usage and severe disease. In vitro studies have demonstrated that vitamin D enhances steroid responsiveness in adult asthmatics. Vitamin D may play an important role in pulmonary health by inhibiting inflammation, in part through maintaining regulatory T cells, and direct induction of innate antimicrobial mechanisms. More research is required to fully understand the role of vitamin D in the maintenance of airway homeostasis and address the diagnostic and therapeutic implications vitamin D may have in the future of asthma management. This review summarises the current understanding and uncertainties regarding the effect of vitamin D deficiency and insufficiency in children with asthma.

Defective IL-10 expression and in vitro steroid-induced IL-17A in paediatric severe therapy-resistant asthma

Background Understanding of immune mechanisms underpinning asthma has emerged from studies in adults. It is increasingly recognised, both immunologically and in the development of novel therapies, that adult responses cannot be used accurately to predict those of children. Methods Using a well-defined paediatric cohort of severe therapy-resistant asthma (STRA) patients, we investigated cytokine profiles in the airway by analysis of bronchoalveolar lavage fluid. The in vitro capacity of peripheral blood mononuclear cells (PBMCs) for cytokine production was also assessed following polyclonal T cell activation in culture, in the absence or presence of dexamethasone and 1α,25-dihydroxyvitamin D3. Results Children with both moderate and STRA had significantly diminished levels of anti-inflammatory interleukin (IL)-10 in airway lavage samples when compared with non-asthmatic controls (p<0.001). Their PBMCs also demonstrated significantly impaired capacity to secrete IL-10 in culture (p<0.001). Dexamethasone regulated the balance between PBMC IL-10 and IL-13 production, increasing IL-10 secretion (p<0.001) and decreasing IL-13 (p<0.001) but unexpectedly enhanced IL-17A production in all groups—most strikingly in the STRA cohort (p<0.001). The inclusion of the active form of vitamin D, 1α,25-dihydroxyvitamin D3, in culture enhanced dexamethasone-induced IL-10 (p<0.05) without marked effects on IL-13 or IL-17A production. Furthermore, systemic vitamin D status directly correlated with airway IL-10 (r=0.6, p<0.01). Conclusions These findings demonstrate reduced peripheral and local IL-10 synthesis in paediatric asthma, and support therapeutic augmentation of low circulating vitamin D in severe, difficult-to-treat asthma, in order to correct impaired IL-10 levels. Conversely, steroids enhanced IL-17A levels, and therefore any steroid-sparing properties of vitamin D may have additional benefit in STRA.

Long-term effectiveness of a staged assessment for paediatric problematic severe asthma

To the Editors: The recently proposed term “problematic severe asthma” (PSA) [1] describes children with persistent symptoms and/or severe exacerbations despite high dose treatment [2]. They suffer significant morbidity and consume a disproportionate amount of healthcare resources. Children with PSA are heterogeneous with respect to lung function [3] and asthma phenotype [4] and warrant careful evaluation. PSA [5] encompasses 1) wrong diagnosis; 2) significant comorbidities; 3) difficult asthma with potentially reversible factors such as poor inhaler technique, ongoing allergen exposure or poor adherence to treatment [6]; and 4) severe therapy-resistant asthma (STRA) that is refractory to maximal treatment despite optimised standard management [7]. PSA children were enrolled in a staged assessment protocol to differentiate difficult asthma from STRA [6]. The different stages were as follows. Stage 1: a detailed re-evaluation of the child including a nurse-led outpatient assessment with evaluation of inhaler device and technique, psychosocial questionnaire, a home and school visit, an assessment of adherence including prescription uptake check and a final multidisciplinary team discussion to decide whether the child had difficult asthma or STRA [6]. Stages 2 and 3: children identified as STRA progressed to invasive investigations including bronchoscopy and assessment of steroid response [7]. Differentiating difficult asthma from STRA is important to avoid unnecessary invasive procedures and side effects from additional treatments such as methotrexate. Furthermore, the healthcare costs of these approaches cannot be justified if simply improving basic management may improve asthma control. There have been no longitudinal studies that have assessed the long-term efficacy of a staged assessment of paediatric PSA. We hypothesised that the stage 1 assessment distinguishes difficult asthma from STRA and results in …

Intraepithelial neutrophils in pediatric severe asthma are associated with better lung function

Background Neutrophils and IL‐17A have been linked mechanistically in models of allergic airways disease and have been associated with asthma severity. However, their role in pediatric asthma is unknown. Objectives We sought to investigate the role of neutrophils and the IL‐17A pathway in mediating pediatric severe therapy‐resistant asthma (STRA). Methods Children with STRA (n = 51; age, 12.6 years; range, 6‐16.3 years) and controls without asthma (n = 15; age, 4.75 years; range, 1.6‐16 years) underwent clinically indicated fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), endobronchial brushings, and biopsy. Neutrophils, IL‐17A, and IL‐17RA–expressing cells and levels of IL‐17A and IL‐22 were quantified in BAL and biopsies and related to clinical features. Primary bronchial epithelial cells were stimulated with IL‐17A and/or IL‐22, with and without budesonide. Results Children with STRA had increased intraepithelial neutrophils, which positively correlated with FEV1 %predicted (r = 0.43; P = .008). Neutrophilhigh patients also had better symptom control, despite lower dose maintenance inhaled steroids. Submucosal neutrophils were not increased in children with STRA. Submucosal and epithelial IL‐17A–positive cells and BAL IL‐17A and IL‐22 levels were similar in children with STRA and controls. However, there were significantly more IL‐17RA–positive cells in the submucosa and epithelium in children with STRA compared with controls (P = .001). Stimulation of primary bronchial epithelial cells with IL‐17A enhanced mRNA expression of IL‐17RA and increased release of IL‐8, even in the presence of budesonide. Conclusions A proportion of children with STRA exhibit increased intraepithelial airway neutrophilia that correlated with better lung function. STRA was also characterized by increased airway IL‐17RA expression. These data suggest a potential beneficial rather than adverse role for neutrophils in pediatric severe asthma pathophysiology.

Genetic testing in children with surfactant dysfunction

Objectives To present the UK experience in genetic diagnoses of surfactant protein dysfunction disorders and develop a referral algorithm for neonates and children with persistent respiratory problems. Materials and methods Between 2006 and 2011, 427 cases were referred for surfactant mutation analyses to the North East Thames Regional Molecular Genetics Laboratory at Great Ormond Street Hospital, London. The results were reviewed and referring physicians of mutation positive cases contacted to complete a questionnaire providing clinical, radiological, histological and outcome information. Results 25 new cases were found to have genetic mutations for surfactant dysfunction disorders (7.5%), with six resulting in surfactant protein B dysfunction, seven surfactant protein C dysfunction and 12 ATP-binding cassette subfamily A member 3 (ABCA3) dysfunction. The referrals were from 15 different paediatric centres. In addition, three affected surfactant protein B (SFTPB) cases were prenatal diagnoses, following the birth of previously affected children. The majority of the confirmed cases (23 of 25) were born after 37 weeks gestation. All children with SFTPB dysfunction and the majority of ABCA3 patients presented with respiratory distress at birth. All SFTPB cases died from intractable respiratory failure. The outcome for ABCA3 mutations was variable with seven survivors. The clinical and radiological presentation of surfactant protein C (SFTPC) patients suggested mainly interstitial lung process with the majority surviving on medication. Conclusions Surfactant mutation analysis is now well established in the UK and allows better genetic diagnosis and counselling. The rarity of the condition makes it difficult to develop a validated algorithm for genetic evaluation with a need for international networking. Referrals need to be rationalised for the service to be time and cost effective.

1α,25-Dihydroxyvitamin D3 promotes CD200 expression by human peripheral and airway-resident T cells

Background CD200, a cell-surface immunoglobulin-like molecule expressed by immune and stromal cells, dampens the pro-inflammatory activity of tissue-resident innate cells via its receptor, CD200R. This interaction appears critical for peripheral immune tolerance, particularly in the airways where excessive inflammation is undesirable. Vitamin D contributes to pulmonary health and promotes regulatory immune pathways, therefore its influence on CD200 and CD200R was investigated. Methods CD200 and CD200R expression were assessed by qPCR and immunoreactivity of human lymphoid, myeloid and epithelial cells following 1α,25-dihydroxyvitamin D3 (1α,25VitD3) exposure in vitro and in peripheral T cells following 1α,25VitD3 oral ingestion in vivo. The effect of 1α25VitD3 was also assessed in human airway-resident cells. Results 1α25VitD3 potently upregulated CD200 on peripheral human CD4+ T cells in vitro, and in vivo there was a trend towards upregulation in healthy, but not asthmatic individuals. CD200R expression was not modulated in any cells studied. CD200 induction was observed to a lesser extent in CD8+ T cells and not in B cells or airway epithelium. T cells isolated from the human airway also responded strongly to 1α25VitD3 to upregulate CD200. Conclusions The capacity of 1α,25-dihydroxyvitamin D3 to induce CD200 expression by peripheral and respiratory tract T cells identifies an additional pathway via which vitamin D can restrain inflammation in the airways to maintain respiratory health.

Survey of the use of non-invasive positive pressure ventilation in UK and Australasian children with cystic fibrosis

Non-invasive positive pressure ventilation (NIPPV) for respiratory failure in cystic fibrosis (CF) is frequently used in adults and has been shown to be of benefit to patients with advanced disease in terms of stabilisation of lung function,1 reduction in symptoms and increased exercise capacity.2 When used as an adjunct to physiotherapy, NIPPV increases oxygen saturations (Spo2), tidal volume, maximum expiratory muscle strength and ease of sputum clearance.3 4 There are no guidelines for the assessment of gas exchange or timing and mode of NIPPV initiation in patients with CF. The British Thoracic Society guideline states ‘there is insufficient evidence to recommend its routine use in patients with CF’.5 The aim of this study was to establish current …