Aspiration lung disease in children with severe neurodisability

Background: Children with severe neurodisability (ND) commonly suffer with respiratory disease and this is the leading cause of premature death. The nature of this respiratory disease is however, poorly understood. The underlying aetiology is often multifactorial, but aspiration (direct or reflux) is likely to play a key role. Unfortunately, diagnostic tests for reflux or direct aspiration are limited. There is a clinical need for high quality research on children with severe ND to define underlying mechanisms of respiratory disease and guide management. Aims: In this study, the aims were to (i) characterise respiratory symptoms and their relationship with lower airway inflammation in children with severe ND, (ii) explore available bronchial lavage (BAL) biomarkers of reflux aspiration, assessing their validity and their relationship to clinical and airway inflammatory data, (iii) develop a novel assay for the accurate detection/quantification of pepsin in BAL, (iv) investigate the validity of an alpha-amylase assay in paediatric BAL and explore the relationship of alpha-amylase levels with lower airway inflammation and clinical symptoms, and (v) investigate the effects of pH alteration and pepsin exposure on airway epithelial cells (AEC). Methods: Clinical data and BAL samples were collected from children with severe ND at times of stability and respiratory deterioration and also from healthy controls. BAL differential cell counts and cytokine measurements (ELISA) were performed. Lower airway microbial colonisation/infection was assessed. BAL pepsin measurement was attempted using a number of methods, and the feasibility of inhibitor affinity enrichment and LC-MRM-MS techniques to identify and quantify BAL pepsin was explored using SDS PAGE gel and mass spectrometry analysis. BAL alpha-amylase activity was measured using an ethylidene-pNP-G7 based assay. BEAS-2B cells were cultured in monolayer and the cytotoxic/inflammatory effects of pH alteration and pepsin exposure were explored through trypan blue staining and cytokine assays. Results: Children with severe ND have burdensome chronic respiratory symptoms that impact on their quality of life and that of their families’. Symptoms may relate to lower airway inflammatory levels. A trend for greater airway neutrophilia and respiratory symptoms was seen in those with lower airway microbial positivity. Available ‘in house’ pepsin ELISA assays were not robust but Western Blot results indicated a higher frequency of BAL pepsin positivity in ND patients with acute respiratory deterioration. Notably, positivity was also found in some healthy controls, highlighting the need for a quantitative assay. The use of an immobilized inhibitor (pepstatin agarose) to recover pepsin from paediatric BAL samples was shown to be feasible. Subsequent digestion of pepsin and detection by LC-MSMS with selective ion monitoring was demonstrated. Significantly increased BAL alpha-amylase levels were seen in Elective-ND patients compared to healthy controls and furthermore, significant correlations were observed with BAL lower airway inflammatory markers. BEAS-2B cells were sensitive to mild acidification, with up-regulation of TGF Beta-1, IL-6 and IL-8 mRNA expression. A corresponding rise in protein secretion was not necessarily seen and in some cases, was significantly down-regulated. No significant cytotoxicity or decrease in cell viability was observed in any condition. Conclusions: In children with severe ND, chronic respiratory symptoms may be directly related to lower airway inflammation and bacterial colonization. Measurement and quantification of pepsin in BAL (as a marker of reflux aspiration) is difficult. However, an inhibitor affinity enhanced mass spectrometry based technique has potential as a ‘gold-standard’ method for identification and quantification of pepsin in BAL. BAL alpha-amylase activity shows promise as a biomarker of direct aspiration. Mild alterations in airway pH may directly contribute to the pathophysiology of inflammatory airway disease and specifically, reflux-aspiration related lung disease in this group. This requires further study as a potential novel therapeutic target.