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Identification and Characterisation of Mucin Abnormalities in the Ganglionic Bowel in Hirschsprung’S Disease

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Identification and Characterisation of Mucin Abnormalities in the Ganglionic Bowel in Hirschsprung’S Disease IDENTIFICATION AND CHARACTERISATION OF MUCIN ABNORMALITIES IN THE GANGLIONIC BOWEL IN HIRSCHSPRUNG’S DISEASE By Ruth Marian Speare Submitted for MD (Res) 1 Abstract Hirschsprung’s disease (HD) is a congenital abnormality of unknown origin, characterised by a lack of ganglion cells in the distal colon which results in functional colonic obstruction. The major cause of morbidity and mortality in these children results from an inflammatory condition called enterocolitis. Mucins are large glycoproteins produced by intestinal cells which are a vital part of the colonic defensive barrier to infection. Previous work has found that this barrier is deficient in children with HD in the aganglionic colon and in the immediately adjacent ganglionic colon and that this is related to the risk of developing enterocolitis. This study aimed to further investigate the mucin defensive barrier in a greater region of the ganglionic colon in HD, to establish the extent of any mucin deficiencies and whether these were confined to a limited region close to the aganglionic colon. Mucosal biopsies were collected at intervals along the colon at the time of corrective surgery or colostomy closure in the controls. Organ culture with radioactive mucin precursors was performed and the mucin produced was purified and analysed, results quantified by DNA content in the sample. Lectin binding studies were also carried out. Patients were found to produce lower levels of new mucins most distally, but much higher levels five centimetres proximal when compared to controls. The rest of the colon studied also showed changes in mucin production, with a lack of production of gel-forming mucins and sulphated secreted mucins in Hirschsprung’s disease higher up the colon. Lectin binding studies, which indicate the presence of existing mucins as well as those produced during organ culture, demonstrated greater levels of binding in patients compared to controls for wheat germ agglutinin and Maackia amurensis agglutinin. 2 CONTENTS Acknowledgements 11 Abbreviations 12 Chapter 1: Introduction - Mucins 1.0: Basic Structure 16 1.1: Polypeptide Structure 20 1.2: Oligosaccharide Structure 25 1.3: Multimetric Structure and Assembly 30 1.4: Role and Function 35 1.5: Alterations in Disease 41 1.6: Alterations in Children 45 Chapter 2: Introduction – Hirschsprung’s Disease and the Enteric Nervous System 2.0: Hirschsprung’s Disease - Historical Background 49 2.1: Hirschsprung’s Disease - Incidence and Aetiology 51 2.2: Hirschsprung’s Disease - Clinical Presentation 52 2.3: Hirschsprung’s Disease – Pathophysiology 53 2.4: The Enteric Nervous System – Structure 54 2.5: The Enteric Nervous System – Function 57 2.6: The Enteric Nervous System - Hirschsprung’s Disease 58 2.7: The Enteric Nervous System – Development 59 2.8: Hirschsprung’s Disease – Pathogenesis 65 2.9: Hirschsprung’s Disease – Treatment 70 2.10: Hirschsprung’s Disease – Outcome 72 2.11: Hirschsprung’s Disease - Transitional Zone 73 2.12: Hirschsprung’s Disease – Enterocolitis 75 2.13: Hirschsprung’s Disease - Mucin Alterations 78 Chapter 3: Materials and Method 3.0: Subject Recruitment and Study Groups 82 3.1: Retrieval of Specimens 83 3.2: Reagents 84 3.3: Organ Culture 87 3.4: Cellular Fractionation 88 3 3.5: Gel Filtration 89 3.6: Determination of Radiolabel Incorporation 90 3.7: DNA Analysis 93 3.8: Slot Blot Analysis 95 3.9: Analysis of Data 105 3.10: Statistical Analysis 109 Chapter 4: Results 4.0: Study Subject Data 110 4.1: Gel Filtration Graphs 112 4.2: Total Mucin Incorporation 115 4.3: Incorporation within the Sub-Fractions of the Mucin Peak 121 4.4: Total Back Peak Incorporation 129 4.5: Incorporation within the Sub-Fractions of the Back Peak 135 4.6: Lectin Binding 143 4.6.1: Wheat Germ Agglutinin Mucin Binding 143 4.6.2: Wheat Germ Agglutinin Binding within 146 Mucin Sub-Fractions 4.6.3: Maackia Amurensis Agglutinin Mucin Binding 147 Chapter 5: Discussion 5.0: Gel Filtration Graphs 148 5.1: Incorporation within the Mucin Peak 151 5.2: Incorporation within the Mucin Peak Sub-Fractions 161 5.3: Incorporation within the Back Peak 168 5.4: Incorporation within the Back Peak Sub-Fractions 171 5.5: Lectin Binding 173 5.5.1: Wheat Germ Agglutinin 174 5.5.2: Correlation between Incorporation and WGA 178 5.5.3: Maackia Amurensis Agglutinin Binding 185 5.6: Limitations of the Study 188 Chapter 6: Conclusions and Suggestions for Further Work 192 References 197 Appendix 232 4 List of Figures 1.1: A schematic representation of a MUC2 subunit showing the 20 'bottle brush' appearance 1.2: A schematic representation of the polypeptide domains of gel- 23 forming mucins 1.3: Difference O-linked oligosaccharide core structures found in 27 mucins 1.4: Metabolic pathways for the generation of nucleotide sugars 32 including sialic acid 2.1: Histology section from a rectal biopsy from normal bowel 53 2.2: Histology section from a rectal biopsy from a Hirschsprung's 53 patient 2.3: Schematic representation of the layers of the intestine showing 55 the nerve plexi 2.4: Histological section showing the layers of the intestine wall and 56 their relation to Auerbach's plexus 2.5: Schematic representation of some of the signaling pathways 63 involved in enteric nervous system development 2.6: Schematic representation of the different types of pull-through 71 procedure for Hirschsprung's disease 3.1: Schematic representation of the distal colon and biopsy sites 83 3.2: Schematic representation of the organ culture equipment 87 3.3: An example of a standard DNA curve 94 3.4: Example of a slot blot obtained using WGA lectin 101 3.5: Example of the densitometry trace from a WGA slot blot 102 3.6: Example of a standard mucin curve obtained from a WGA slot 103 blot 3.7: Example of a standard mucin curve obtained from a MAA slot 104 blot 3.8: Example of a patient graph of gel filtration fraction number and 106 incorporation 5 3.9: Example of a control graph of gel filtration fraction number and 106 incorporation 4.1: Example patient graph showing mucin and back peaks 114 4.2: Example control graph showing mucin and back peaks 114 4.3: Individual subject graphs of total mucin peak [3H]-glucosamine 117 incorporation at colonic points A to D 4.4: Individual subject graphs of total mucin peak [35S]-sulphate 119 incorporation at colonic points A to D 4.5: Scatter graph of mucin peak [3H] incorporation 120 4.6: Scatter graph of mucin peak [35S] incorporation 120 4.7: Mean ratio of [35S]/[3H] incorporation within SSF mucin peak 128 4.8: Mean ratio of [35S]/[3H] incorporation within CSF mucin peak 128 4.9: Mean ratio of [35S]/[3H] incorporation within CIF mucin peak 128 4.10: Individual subject graphs of total back peak [3H]-glucosamine 131 incorporation at colonic points A to D 4.11: Individual subject graphs of total back peak [35S]-sulphate 133 incorporation at colonic points A to D 4.12: Scatter graph of back peak [3H] incorporation 134 4.13: Scatter graph of back peak [35S] incorporation 134 4.14: Mean ratio [35S]/[3H] incorporation within SSF back peak 142 4.15: Mean ratio [35S]/[3H] incorporation within CSF back peak 142 4.16: Mean ratio [35S]/[3H] incorporation within CIF back peak 142 4.17: Mean [35S] incorporation within CIF fraction of back peak 143 4.18: Individual subject graphs of total mucin peak WGA binding at 145 colonic points A to D 4.19: Scatter graph for MAA binding 147 5.1: Example of [3H]-glucosamine incorporation gel filtration graph 148 5.2: Example of [35S]-sulphate incorporation gel filtration graph 149 5.3: Example of patient [3H]-glucosamine incorporation gel filtration 150 back peak graph 5.4: Example of control [3H]-glucosamine incorporation gel filtration 150 back peak graph 5.5: Control mean mucin [3H] incorporation A-D 153 6 5.6: Control mean mucin [35S] incorporation A-D 153 5.7: Ratio control mean [35S]/[3H] incorporation 154 5.8: Patient mean mucin [3H] incorporation A-D 154 5.9: Patient and control mean mucin [35S] incorporation A-D 155 5.10: Ratio patient mean [35S]/[3H] incorporation 155 5.11: Patient Subgroup Mean [3H] Incorporation A-D 157 5.12: Controls and Patient Subgroups Mean [3H] Incorporation 158 5.13: Controls and Patient Subgroups Mean [35S] Incorporation 158 5.14: Scatter Graph of Individual Patients [3H]-glucosamine 159 Incorporation at Colonic Point B 5.15: Controls and Patient Subgroups Mean [3H] Incorporation 161 5.16: Mean control [3H] incorporation A-D within the three mucin 163 containing fractions 5.17: Mean patient [3H] incorporation A-D within the three mucin 163 containing fractions 5.18: Mean [3H] incorporation A-D within the CIF mucin fraction 164 5.19: Mean [3H] incorporation A-D within the SSF mucin fraction 165 5.20: Proportion of [35S] incorporation within the mucin fractions at A 166 and D 5.21: Mean ratio [35S]/[3H] incorporation within the SSF mucin 167 fraction 5.22: Mean control [3H] incorporation A-D in back peak 169 5.23: Mean [3H] incorporation within SSF fraction of back peak 172 5.24: Proportion of [3H] incorporation within control back peak 172 fractions 5.25: Scatter graph of WGA mucin binding 174 5.26: Mean WGA binding within the mucin peak 175 5.27: Mean WGA binding within low patient subgroup 176 5.28: Mean WGA binding within SSF fraction 177 5.29: Mean WGA binding within CIF fraction 178 5.30: Mean Patient [3H] Incorporation A-D 179 5.31: Mean Patient WGA Binding A-D 179 7 5.32: Mean Patient [35S] Incorporation A-D 179 5.33: Mean Control [3H] Incorporation A-D 181 5.34: Mean Control WGA Binding A-D 181 5.35: Mean Control [35S] Incorporation A-D 181 5.36: Mean
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