Pepsin as biomarkers of reflux

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Pepsins as biomarkers Intermittent reflux of gastric juice into the oesophagus and beyond is a normal physiological event. Consequently tissues such as the oesophagus, larynx, nasopharynx and airways must have mechanisms of mucosal protection in place. Currently attempts to measure reflux in humans use invasive diagnostic tools such as 24 hour pH monitoring or impedance measurement, both techniques involve placing a thin walled tube through the nostril into the oesophagus for a distance of about 45 cm. Therefore in order to determine reflux levels a biomarker needs to be identified and quantified. Pepsins, a major enzyme component of gastric juice (present at 0.2-1mg/ml) are such biomarkers. The aims of this project are two fold; (1) To define the normal levels of reflux and develop a simple assay system for pepsin that can be developed commercially. (2) To characterise tissue specific protection mechanisms. At present no commercial assay is available to measure pepsin in biological fluids and tissues and a central part of this study will be to develop such an assay with Prof Pearson at Newcastle University and Technostics, an SME based in Kingston upon Hull, East Yorkshire. To date there is little or no information in the literature to define the normal levels of reflux or the protective mechanisms present in these tissues. Mucus, bicarbonate, differential expression of carbonic anhydrase isoenzymes, stress proteins and cell adhesion molecules have all been implicated. In ICaMB at Newcastle University the student will use in vitro cell and explant culture techniques to investigate the effects of pepsin on mucosal integrity in the oesophagus, larynx, nasopharynx and the airways. Techniques employed will include immunohistochemistry, quantitative PCR, ELISA and multiplex techniques to measure mucins, carbonic anhydrase isoenzymes, epithelial stress proteins and cell adhesion molecules. While at Technostics the student will be trained in all aspects of commercial assay development including antibody design using computer programs to identify key epitopes within the pepsin protein; monoclonal antibody production; lateral flow dipstick technology. Assay development including assessing reproducibility, reliability and ease of use in the clinical setting will be carried out at both Technostics and Newcastle University. This will involve the student interacting with industrial and clinical colleagues. Thus the student will be exposed to a wide range of techniques in commercial and academic environments. This pepsin assay when fully developed and validated will identify reflux events by the appearance of pepsin in sputum expectorated by the subject and in a dip stick format could be used in the doctors surgery. It will also have applications in disease diagnosis and directing treatment management, where excessive reflux can lead to tissue damage in the aero-digestive tract. The need for such an assay is well demonstrated by the recent identification of health problems associated with reflux. We and others have demonstrated reflux of gastric juice is implicated in the development of several upper airway diseases e.g. rhinitis, sinusitis, otitis media with effusion, voice disorders, globus and laryngeal cancer and several lung diseases e.g. asthma and COPD. We have recently demonstrated a key role for reflux in lung transplant rejection. Consequently understanding the nature of the refluxate and how tissues defend themselves are critical questions. Also medical practise is crying out for a simple, easy to use diagnostic tool for reflux.