Neutrophil Activation in Inflammatory Bowel Disease

Abstract Inflammatory Bowel Disease (IBD) is an inflammatory condition in which neutrophils play an important role. Colonoscopy with biopsy is thought to be the best method for evaluating inflammation location, extent, and severity. However, the invasiveness of endoscopic examinations represents a strong limitation to their frequent use. Various studies have described faecal markers as powerful biomarkers of inflammation of the intestinal mucosa in patients with IBD. This thesis details the use of calprotectin and myeloperoxidase (MPO) as biomarkers of disease severity in patients with IBD, and aims to assess whether MPO is a superior biomarker to calprotectin. Abundant levels of both calprotectin and MPO are found in neutrophils. A total of 500 patients were recruited into the evaluation of Novel Biomarkers in Inflammatory Bowel Disease project cohort. However, only 100 of the initial stool samples from patients with or without IBD were used for this research. The samples had been stored at -80°C for up to one year. A short extraction procedure using 100 mg of faeces and approximately 4.9 mL of the appropriate extraction buffer was carried out. Levels of calprotectin and MPO protein were then measured by sandwich enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of MPO was also measured using 3,3’,5,5’-tetramethylbenzidine (TMB) as a reducing substrate. TMB forms a blue product when it reacts with peroxidase enzymes such as MPO. The resulting colour change was read on a microplate reader at a wavelength of 630 nm. Levels of calprotectin, MPO protein, and MPO TMB activity were significantly higher in IBD patients compared to controls. There were significant correlations between calprotectin, MPO protein and MPO TMB activity (p < 0.001). Levels of calprotectin and MPO protein correlated significantly with endoscopic disease severity in patients with CD (r = 0.487, p = 0.001, n = 41, r = 0.483, p = 0.001, n = 41, respectively) and UC (r = 0.677, p << 0.001, n = 35, r = 0.552, p < 0.001, n = 35, respectively). Consequently, both calprotectin and MPO protein were able to discriminate between IBD patients with inactive and high disease severity. However, MPO TMB activity failed to correlate with disease severity in CD and UC patients (r = 0.303, p = 0.054, n = 41; r = 0.258, p = 0.134, n = 35, respectively). The results obtained from this research show that calprotectin and MPO protein correlate strongly with each other. There was also a strong correlation between MPO protein and disease severity, and MPO could successfully distinguish between inactive and high disease severity in CD and UC patients. This suggests that MPO may be useful in the diagnosis and follow-up of patients with IBD. Although, the relationships between MPO TMB activity and disease severity in patients with CD and UC were not significant results were still very promising. This assay could prove to be a faster and more cost effective approach to aid in the diagnosis and follow up of patients with IBD in the future. However, further development and optimisation of the MPO ELISA and MPO TMB assay is required to validate the results from this research