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Title: Investigating pathogenesis of reflux aspiration
Author: Hackett, A. P.
ISNI:       0000 0004 6425 1829
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Background: Reflux aspiration, the chronic microaspiration of aerosolised gastric contents, is a common cause of morbidity and mortality in children and adults with severe neurodisability. It is believed that reflux aspiration causes a chronic lowering of airway pH leading to recurrent respiratory tract infections, inflammation and fibrosis. Previous, in vitro and in vivo studies show exposure to very low pH causes airway inflammation, but are not physiologically relevant to chronic microaspiration where the airways are only mildly acidic. The effects of chronic dilute gastric juice exposure on airway epithelium has not been reported. Diagnosis of reflux aspiration is difficult. No objective, sensitive and specific test has been clinically validated. Although pepsin has been suggested as an ideal biomarker for reflux aspiration, published methods for detecting airway pepsin lack the specificity or sensitivity required for widespread clinical use. Aims: Firstly, to assess the effects of mild pH alteration on airway epithelial cells. Secondly, to assess the effects of dilute gastric juice on airway epithelial cells. Thirdly, to develop an effective assay for reflux aspiration using pepsin as a biomarker. Methods: For the first aim, BEAS-2B airway epithelial cells (AECs) were exposed to weakly acidic pH (pH5.5 – 6.5) alone, in the presence of lipopolysaccharide (LPS) or prior to LPS stimulation. Interleukin-6 (IL-6) and IL-8 expression was measured by quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). For aim 2, AECs were repeatedly exposed to dilute gastric juice for 8 hours a day for 5 days. IL-6, IL-8 and transforming growth factor-ß (TGF-ß) gene and protein expression was measured by qPCR and ELISA and expression of cell phenotype markers E-cadherin, Vimentin and Fibronectin by qPCR. For aim 3, antibodies were tested for pepsin specificity using a modified western blot protocol. An antibody was selected for assay development using pepstatin beads and the Wes capillary western system. The ability of the assay to identify pepsin in bronchoalveolar lavage fluid (BAL) was then assessed. Results: Incubation in weakly acidic media for long periods (24 hours) is cytotoxic to AECs but does not induce IL-6 or IL-8 expression. LPS-induced IL-6 and IL-8 expression is reduced when AECs are stimulated in a weakly acidic environment. Chronic exposure to dilute gastric juice induced IL-6 and IL-8 but not TGF-ß expression. Exposure did not induce biologically significant changes in E-cadherin, Vimentin or Fibronectin expression. Pepsin could be detected by Wes at low concentrations. Commercially available anti-pepsin antibodies non-specifically bind other BAL proteins thus pepstatin beads were required to affinity purify pepsin. Recovery rates of pepsin from pepstatin beads were very low but could be detected on Wes. Conclusions: A weakly acidic environment does not induce AEC inflammatory marker expression but decreases the inflammatory responses to bacterial endotoxin suggesting a reduced response to infection. Chronic exposure to dilute gastric juice does induce expression of inflammatory cytokines but does not induce fibrosis through EpithelialMesenchymal Transition. The pepstatin bead – Wes based assay whilst effective at detecting pepsin requires further development and is limited by the lack of high affinity antibodies.
Supervisor: McNamara, P. S. ; Flanagan, B. F. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral