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Moftah Hussin Alhagamhmad Discovering the mechanism of action of nutritional therapy and evolving an innovative formula for the treatment of Crohn’s disease A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Moftah Hussin Alhagamhmad School of Women’s and Children’s health Faculty of Medicine The University of New South Wales August 2015 Table of contents List of abbreviations …………………………………………………………………………………………..V List of Figures……………………………………………………………………………………………...... IX List of Tables…………………………………………………………………………………………….......XII Publications………………………………………………………………………………………………....XIII Acknowledgements........................................................................................................................................XIV Chapter 1: Literature review and aims 1.1 Background and disease discovery………………………………………………………………………..1 1.2 Epidemiology…………………………………………………………………………………………..….2 1.3 Pathophysiology of Crohn’s disease 1.3.1 Alteration in the immune response…………………………………………………………….….6 1.3.2 Role of bacteria…………………………………………………………………………………..13 1.3.3 Role of genetics………………………………………………………………………………….26 1.3.4 Role of environmental factors…………………………………………………………………...28 1.4 Diagnosis of Crohn’s disease 1.4.1 History and clinical examination………………………………………………………………..33 1.4.2 Laboratory findings……………………………………………………………………………..33 1.4.3 Imaging studies& endoscopic evaluation……………………………………………………….34 1.4.4 Crohn’s disease activity index…………………………………………………………………...34 1.5 Symptomatology of Crohn’s disease 1.5.1 Intestinal Manifestations…………………………………………………………………………36 1.5.2 Extra-intestinal manifestations…………………………………………………………………..36 1.6 Management of Crohn’s disease 1.6.1 Corticosteroids Medications……………………………………………………………………...42 1.6.2 Immunomodulators……………………………………………………………………………….44 1.6.3 AminoSalcylic Acid drugs………………………………………………………….…………….46 1.6.4 Biological agents…………………………………………………………………….…………...48 1.6.5 Antibiotics……………………………………………………………………………….……….51 1.6.6 Probiotics and prebiotics………………………………………………………….……………...53 1.6.7 Surgery…………………………………………………………………………………………...55 1.7 Role of nutritional therapy in management of Crohn’s disease 1.7.1 Overview………………………………………………………………………………………….57 1.7.2Mechanisms of action of exclusive enteral nutrition (EEN).……...………………………………61 1.7.3 Nutritional benefits of diet therapy………………………………………………………………..74 1.7.4 EEN as a treatment option………………………………………………………………………...77 1.8 Novel nutritional treatments 1.8.1 Glutamine……………………………………………………………………………………...….93 1.8.2 Arginine……………………………………………………………………………………….…..96 1.8.3 Curcumin………………………………………………………………………………………….98 1.8.4 Omega-3 fatty acids……………………………………………………………………………...102 1.8.5 Vitamin D3……………………………………………………………………………………....104 1.9 Project aims and hypotheses ……………………………………………………………………………105 Chapter 2: Methodology 2.1 In Vitro model of IBD 2.1.1 Cell culture and induction of inflammation………………………………………………………107 2.1.2 Cell viability assessment………………………………………………………….……..……….115 2.1.3 Enzyme Linked Immunosorbent assay…………………………………………………………...119 2.1.4 Western blot…………………………………………………………………….……………...…122 2.1.5 RNA extraction and amplification by Real-Time PCR…………………………………………..128 2.1.6 Single- Labelling Immunofluorescence…………………………………………………………...132 2.1.7 Kinase Assay…………………………….………………………………………………………..134 2.1.8 Affymetrix assay………………………………………………………………………………….136 2.2 In vivo models of IBD 2.2.1 Background…………………………………….…………………………………………………..138 2.2.2 Mice strain and acclimatisation…………………………………………………………………....141 2.2.3 Experimental protocols and induction of colitis…………………………………………………...142 2.2.4 Treatment composition and preparation…………………………………………………..……….145 2.2.5 Evaluation of colitis and response to treatment…………………………………………………....147 2.3 Ex-Vivo human cultured colonic biopsies 2.3.1 Introduction…………………………………………………………………………………….….152 2.3.2 Participants and inclusion/exclusion criteria………………………………………………………153 2.3.3 Endoscopy and histology examination…………………………………………………………….154 2.3.4 Biopsy culture and treatment………………………………………………………………………155 2.3.5 Cytokines immunoassay and LDH activity………………………………………………………..156 Chapter 3: Investigating the anti-inflammatory properties of PF and elucidating the mechanisms in vitro. 3.1 Introduction……….……….…………………..……………………………………………………158 3.2 Hypothesis...................................................................................................................................…...160 3.3 Aims………………………………………………………………………………………………....160 3.4 Results…………………………………………………………………………………………...….161 3.5 Discussion………………………………………………………………………...…………………214 3.6 Conclusion………………………………………………………………..…………………………232 Chapter 4: Developing a novel nutritional therapy with enhanced anti-inflammatory properties for CD in vitro. 4.1 Introduction………………………………………………………………………………………....233 4.2 Hypothesis …………………………………………………………………………………….…... 234 4.3 Aims............................................................................................................................................…....234 4.4 Results………………………………………………………………………………………………236 4.5 Discussion…………………………………………………………………………………….…..…255 4.6 Conclusion…………………………………………………………………………………………..262 Chapter 5: Exploring efficacy of the novel nutritional therapy in murine model of colitis 5.1 Introduction ……………………………………………….……………………………………….263 5.2 Hypothesis..................................................................................................................................…...263 5.3 Aims…….……….………………………………………………………………………………….263 5.4 Results……………………………………………………..……………………………………….265 5.5 Discussion…………………………………………………………………………………………..284 5.6 Conclusion………………………………………………………………………………………….292 Chapter 6: Using ex-vivo cultured colonic biopsies from Crohn’s patient to investigate effect of the novel nutritional therapy 6.1 Introduction………………………………….………………………………………………….…..293 6.2 Hypothesis…….……………………………………….…………………………………………....294 6.3 Aims…..….………………………………………………………………………………………….294 6.4 Results.……………………………………………………………………………………………...295 6.5 Discussion……….……………………………………………………………………………….….302 6.6 Conclusion…….…………………………………………………………………………………….305 Chapter 7: Final discussion and conclusions……………………………………………………………..306 Bibliography ………………………………………………………………………………………………..315 List of Abbreviations ALA: Alpha-linolenic acid ANOVA: Analysis of variance 5-ASA: 5-Aminosalicylates ASCA: Anti-Saccharomyces cerevisiae antibodies ATG16L1: autophagy-related 16-like 1 AZA: Azathioprine BCA: Bicinchoninic acid BME: Basal Medium Eagle BMI: Body mass index BSA: Bovine serum albumin CARD15: Caspase activation recruitment domainfamily-15 CBC: Complete blood count CD: Crohn’s disease CDED: Crohn’s disease exclusion diet CDAI: Crohn's disease activity index cDNA: complementary DNA CE: Capsule endoscopy CEACAM: Carcinoembryonic antigen-related cell adhesion molecule COX-2: cyclooxgenase-2 CRP: C-reactive protein CT: Threshold cycle DAPI: 4', 6-diamidino-2-phenylindole DEG: Differentially expressed gene DGGE: Denaturing gradient gel electrophoresis DHA: Docosahexaenoic acid DIA: Disease activity index DMSO: Dimethyl sulfoxide DSS: Dextran sodium sulfate ECCO: European Crohn's and Colitis Organization E: Enterography ESPGHAN: European Society of Pediatric Gastroenterology, Hepatology and Nutrition EEN: Exclusive enteral nutrition EF: Elemental formula EMSA: Electro-mobility shift assay technique EPA: Epicosapentanenoic acid ESR: Erythrocyte sedimentation rate F: Faecalibacterium Ȧ -3 FAs: Omega-3 fatty acids FBS: Fetal bovine serum FDR: False discovery rate adjusted P value FL: Fecal Lactoferrin GH: Growth hormone GIT: Gastro-intestinal tract HLH: Helix-loop-helix H2SO4: Sulphuric acid HRP: Horseradish peroxidase IBD: Inflammatory bowel disease IBDU: IBD unclassified IFN: Interferon IG: Immunoglobulin IGF: Insulin like growth factor Iț%: Inhibitor of kappa B ,țț,ț%NLQDVH IL: Interleukin IRAK: IL-1 receptor associated kinase LCT: Long chain triglycerides LDH: Lactate dehydrogenase LPS: Lipopolysaccharide LZ: Leucine zipper MAP: Mycobacterium Paratuberculosis MAPK: Mitogen activated protein kinases MCP: Monocyte chemoattractant protein MDP: Muramyl dipeptide MEKK1: Mitogen-activated protein kinase kinase kinase-1 MEM: Minimum Essential Medium mM: Millimole MLCK: Myosin II regulatory light chain kinase 6-MP: 6-Mercaptopurine MPO: Myloperoxidase MTT: (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; Thiazolyl blue) MTX: Methotrexate NEAA: Non-Essential Amino Acids Solution NDS: Normal donkey serum NF-ț%1XFOHDUIDFWRU 1) -ț% NO: Nitric Oxide NOD2: Oligomerization domain-2 OTU: Operational taxonomic unit PBMS: Peripheral mononuclear cells PBS: Phosphate Buffered Saline PCDAI: Paediatric Crohn’s disease activity Index PF: Polymeric formula PGA: Physician global assessment tool PSC: Primary sclerosing cholangitis PUFA: Polyunsaturated fatty acid PVDF: Polyvinylidene difluoride RAGE: receptor for advanced glycation end products RMA: Multi-array average RT-PCR: Real time polymerase chain reaction SCFA: Short chain fatty acid TBN: Total parenteral nutrition TGF-ß: Transforming growth factor beta TGGE: Temperature gradient gel electrophoresis TIRAP: TLR associated protein TLR: Toll like receptors TMB: 3, 3’, 5, 5’-Tetramethylbenzidine TNBS: Trinitrobenzene sulfonic acid TNF-Į7XPRXUQHFURVLVIDFWRU-alpha TRAF: TNF-receptor associated factor UC: Ulcerative colitis WR: Working reagent List of Figures 1.1 Aetiology of malnutrition in patients with CD………………………………………………………...….41 2.1 TNBS and DSS colitis model
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