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Title: The role of ABCB1 and ABCB5 in paediatric inflammatory bowel disease
Author: Konidari, Anastasia
ISNI:       0000 0004 8506 3827
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2020
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ABC transporters play an important role in drug disposition. ABCB1, the most studied representative member of this transporter superfamily, can influence drug response and shape multi-drug resistance in cancer and various diseases including inflammatory bowel disease. Adult studies have shown inter-individual variability in expression between patients with inflammatory bowel disease and healthy controls, which can be modified by corticosteroid administration, a common treatment agent for inflammatory bowel disease. ABCB5 is a novel transporter highly homologous to ABCB1. This thesis focusses on an inception pilot cohort of children and young people with newly diagnosed inflammatory bowel disease and healthy controls. The purpose of this thesis is to investigate the expression of ABCB1 and ABCB5 in vitro and ex vivo, to test the hypothesis that both are important in children with inflammatory bowel disease with respect to expression, to investigate how expression varies with genotype, and to explore the possible role of ABCB5 as corticosteroid transporter. De novo production of stable and transient overexpressing mammalian ABCB5 clones confirmed high gene expression at the mRNA level, but no ABCB5 protein isoforms were detectable, despite repeated attempts and investigation of human malignant cell derived ABCB5 expressing clones donated by experts in the field. Radiolabeled drug uptake studies with corticosteroids commonly implemented in the treatment of inflammatory bowel disease, showed that corticosteroid efflux was not mediated by any of the ABCB5 overexpressing mammalian cell lines. Children with newly diagnosed inflammatory bowel disease and healthy controls recruited at a regional tertiary children’s hospital were genotyped for common ABCB1 and novel ABCB5 single nucleotide polymorphisms. The genotype variation observed between cases and controls was not statistically different at the point of diagnosis. The minor allele frequency for rs2032582 (G2677T in exon 21 of the ABCB1 gene) was significantly different between cases and controls (p=0.01), but this did not withstand correction for multiple testing. ABCB1 and ABCB5 gene expression profiling of blood and intestinal biopsies from 16 patients with inflammatory bowel disease and 20 controls was determined. No significant variability was observed in ABCB1 gene expression across ABCB1 genotypes or clinical phenotypes (inflammatory bowel disease versus healthy state). ABCB5 expression was not detectable in blood or intestinal biopsies taken from the small intestine and sigmoid colon of the study participants. ABCB1 and ABCB5 protein expression was characterized by immunohistochemistry in intestinal biopsies from study participants. ABCB1 showed inter and intraindividual variability in expression across blood samples and intestinal biopsies from cases and controls, but this was not significantly different. There was significant correlation between gene expression and protein expression in sigmoid colon (p=0.007). Non-specific background staining for ABCB5 by immunohistochemistry was noted, which did not allow for reliable quantification of ABCB5 protein expression in the small intestine and sigmoid colon. Treatment response to corticosteroids in a subgroup of patients with ulcerative colitis showed weak correlation with ABCB1 expression. In conclusion, in this pilot set of experiments, no difference was identified in ABCB1 and ABCB5 expression at mRNA and protein level in peripheral blood cells, small intestine and colonic biopsies, taken from children with inflammatory bowel disease and healthy controls. The study was limited by the small sample size; further studies will be needed for confirmation of these results. Importantly, there was no ABCB5 expression at the protein level and no functional activity was identifiable with respect to corticosteroid efflux in transfected cell lines. Further work is required to appreciate the role of ABCB5 in intestinal physiology and pathology.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral