published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

潰瘍性結腸炎 (UC) 是一種原因不明的炎症性腸道疾病，治療原則是減輕炎症，但UC的病因有多種，一般消炎藥物如柳氮磺胺吡啶 (sulfasalazine) 等治療都是單靶向並有嚴重副作用，故副作用低、多靶向藥物是必要的。 / Cathelicidin 是一種抗菌抗炎的肽。事實上，鼠的 cathelicidin (mCRAMP) 直腸給藥能緩解小鼠 UC。為了提高療效及方便給藥，mCRAMP 編碼被導入乳酸乳球菌中。乳酸乳球菌是一種能抵抗胃酸的乳酸益生菌，因此口服亦能生產及傳送 cathelcidin 到大腸。 / 小鼠用含3% 葡聚醣硫酸鈉 (DSS) 的水7天以誘導UC。小鼠隨機分為十組，各接受每日一次的口服製劑：(1) 水，(2) DSS，(3, 4) DSS + 10¹° cfu 有或沒有 nisin 誘導的乳酸乳球菌，(5-8) DSS + 10⁸ 或 10¹° cfu有 (N4I) 或沒有 nisin 誘導的 mCRAMP 編碼乳酸乳球菌，(9) DSS + 0.5% 羧甲基纖維素鈉 (CMC-Na) 及 (10) DSS + 600 mg/kg懸浮於0.5% CMC-Na 的 sulfasalazine。 / 研究對 UC 預防效果時，小鼠同時接受 DSS 及治療。所有益生菌製劑中，只有 N4I 能降低中性粒細胞浸潤、脂質過氧化和炎症細胞因子表達，同時保護腸隱窩及黏膜分泌層結構，減少細胞凋亡及腸道菌群。相比之下，sulfasalazine 能抑制炎症但不能阻止結腸結構損傷。 / 進一步研究治療效果時，小鼠在炎症形成後接受四天治療。N4I 能促進結腸黏膜恢復，改善結腸和黏液分泌層的結構。這些作用可能通過刺激細胞增殖和抑制凋亡造成。相對地，sulfasalazine 對結腸組織重組沒有影響。 / 為了研究 mCRAMP 直接消炎作用，小鼠巨噬細胞 RAW 264.7 被脂磷壁酸和脂多醣刺激以模仿 UC 時細菌引起的炎症。mCRAMP 能減輕腫瘤壞死因子-α分泌及IκBα磷酸化並抑制核因子-κB (NF-κB) 活化，炎症酶如誘導型一氧化氮合酶和環氧合酶-2的表達也減少了。mCRAMP可能直接抑制細菌毒素與受體結合和/或直接抑制 NF-κB 產生消炎作用。 / 在研究 mCRAMP 修復黏膜的作用中，證實 mCRAMP 通過 G 蛋白偶聯受體依賴途徑和間接激活表皮生長因子受體、激活下游絲裂原活化蛋白激酶而促進細胞遷移、加速癒合。 / 總括而言，本研究首次顯示mCRAMP編碼乳酸乳球菌對 UC 有保護和治療作用，其抗炎、抗菌及促進黏膜修復作用來自乳酸乳球菌分泌的mCRAMP。多靶向的mCRAMP編碼乳酸乳球菌具有很大潛力，是一種比標準藥物 sulfasalazine 更好的治療結腸炎製劑。 / Ulcerative colitis (UC) is an idiopathic inflammatory bowel disease (IBD). The mainstay of drug treatment is to relieve inflammation. However the aetiology of UC is multi-factorial while most of the anti-inflammatory drugs, such as sulfasalazine, aim at single target with severe side effects. Therefore, a multi-targeted drug with low systemic toxicity is warranted. / Cathelicidin, a host defense peptide, shows anti-microbial and anti-inflammatory effects. Indeed intra-rectal administration of mouse cathelicidin (mCRAMP) alleviated murine colitis. To improve therapeutic efficacy and reduce inconvenience of administration, Lactococcus lactis (L. lactis) was constructed to encode cathelicidin. L. lactis is a lactic acid probiotic which could resist gastric acid and be able to produce and deliver cathelicidin to the colon when given orally. / Murine colitis was induced by 3% dextran sulphate sodium (DSS) given in drinking water for 7 days. Mice were given intragastrically with the following preparations once daily: (1) water, (2) DSS, (3, 4) DSS + 10¹° cfu L. lactis with or without nisin induction, (5-8) DSS + 10⁸ or 10¹° cfu mCRAMP-encoding L. lactis with (N4I) or without nisin induction, (9) DSS + 0.5% sodium carboxymethylcellulose (CMC-Na) and (10) DSS + 600 mg/kg sulfasalazine suspended in 0.5 % CMC-Na. / To study the preventive effects, mice received the above treatments together with DSS administration. N4I but not the other probiotic preparations suppressed inflammation by reducing neutrophil infiltration, lipid peroxidation and inflammatory cytokines expressions. Crypt structure and mucus-secreting layer were conserved together with the reduction of apoptosis and intestinal microbiota. In contrast, sulfasalazine could only suppress inflammation but not the destruction of colonic structure. / To further examine the therapeutic effects, mice received treatments for 4 consecutive days after the inflammation formation. Similarly, only N4I promoted colonic mucosal recovery and preserved colon structure and mucus-secreting layer. These actions are likely mediated through cell proliferation stimulation and apoptosis suppression. Again, sulfasalazine had no effects on colon tissue reconstitution. / The direct anti-inflammatory action of mCRAMP was also studied. Mouse macrophage RAW 264.7 cells were stimulated by lipoteichoic acid and lipopolysaccharide to mimic bacteria-induced inflammation during UC. mCRAMP prevented tumour necrosis factor-α secretion and IκBα phosphorylation followed by nuclear factor-κB (NF-κB) suppression. The inflammatory enzymes including inducible nitric oxide synthase and cyclooxygenase-2 were also reduced. It was postulated that mCRAMP might directly interact with the bacterial toxins to reduce receptor complex binding and/or reduce NF-κB suppression in macrophages. / The repairing action of mCRAMP on mucosal damage was studied in mouse colon cells. mCRAMP incubation reduced the wound size by promoting cell migration through the G-protein coupled receptor and epidermal growth factor receptor transactivation followed by the mitogen-activated protein kinases activation. / In conclusion, the present study demonstrates for the first time the protective and therapeutic roles of mCRAMP-encoding L. lactis in UC. It was the mCRAMP secreted from the probiotic to produce both anti-inflammatory and anti-bacterial actions and further promote mucosal repair. mCRAMP-encoding L. lactis is a multi-targeted agent for IBD. It has a great potential to be a new therapeutic agent better than sulfasalazine for the treatment of UC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Ching Man. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 227-250). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Table of Content --- p.vi / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ulcerative Colitis --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.2 / Chapter 1.1.3 --- Diagnosis --- p.2 / Chapter 1.1.3.1 --- Clinical Presentation --- p.2 / Chapter 1.1.3.2 --- Apparative Diagnostics --- p.3 / Chapter 1.1.3.3 --- Innovative Diagnostics in IBD --- p.4 / Chapter 1.1.4 --- Etiopathogenesis --- p.4 / Chapter 1.1.4.1 --- Genetic Predisposition --- p.4 / Chapter 1.1.4.2 --- Environmental Factors --- p.5 / Chapter 1.1.4.2.1 --- Life Style --- p.5 / Chapter 1.1.4.2.1.1 --- Smoking --- p.5 / Chapter 1.1.4.2.1.2 --- Diet --- p.6 / Chapter 1.1.4.2.1.3 --- Hygiene --- p.6 / Chapter 1.1.4.2.1.4 --- Psychological Stress --- p.6 / Chapter 1.1.4.2.1.5 --- Appendectomy --- p.7 / Chapter 1.1.4.2.2 --- Colonic Mucus --- p.7 / Chapter 1.1.4.2.3 --- Non-steroidal Anti-inflammatory Drugs (NSAIDs) --- p.7 / Chapter 1.1.4.3 --- Alteration of Intestinal Microbiota --- p.8 / Chapter 1.1.4.4 --- Immune Factors --- p.10 / Chapter 1.1.5 --- Existing Treatments --- p.10 / Chapter 1.1.5.1 --- 5-Aminosalicyclic Acid --- p.11 / Chapter 1.1.5.2 --- Corticosteroids --- p.12 / Chapter 1.1.5.3 --- Immunomodulators --- p.12 / Chapter 1.1.5.4 --- Surgical Management --- p.13 / Chapter 1.1.6 --- Emerging Treatments --- p.14 / Chapter 1.1.6.1 --- Antibiotics --- p.14 / Chapter 1.1.6.2 --- Probiotics --- p.14 / Chapter 1.1.6.3 --- Nicotine Patches --- p.14 / Chapter 1.1.6.4 --- Butyrate --- p.14 / Chapter 1.1.6.5 --- Biological Therapies --- p.15 / Chapter 1.1.7 --- Risk of Colorectal Cancer --- p.17 / Chapter 1.2 --- Cathelicidin --- p.17 / Chapter 1.2.1 --- Cathelicidin Family --- p.17 / Chapter 1.2.2 --- Actions and Possible Mechanisms --- p.19 / Chapter 1.2.3 --- Cathelicidin in Ulcerative Colitis --- p.20 / Chapter 1.3 --- Probiotics --- p.21 / Chapter 1.3.1 --- Lactic Acid Bacteria --- p.21 / Chapter 1.3.2 --- Definition of Probiotics --- p.22 / Chapter 1.3.3 --- Possible Mechanisms of Action of Probiotics --- p.23 / Chapter 1.3.3.1 --- Mucous Layer --- p.23 / Chapter 1.3.3.2 --- Host Cell Antimicrobial Peptides --- p.25 / Chapter 1.3.3.3 --- Probiotic Antimicrobial Factors --- p.25 / Chapter 1.3.3.4 --- Epithelial Adherence --- p.27 / Chapter 1.3.4 --- Recent Findings in Ulcerative Colitis Treatment --- p.28 / Chapter 1.4 --- Lactococcus lactis --- p.29 / Chapter 1.4.1 --- Overview --- p.29 / Chapter 1.4.2 --- Gene Expression System --- p.30 / Chapter 1.4.3 --- Nisin-Inducible Controlled Gene Expression (NICE) System --- p.31 / Chapter 1.4.4 --- Recent Studies of Treating Ulcerative Colitis with L. lactis and Recombinant L. lactis --- p.32 / Chapter 1.4.5 --- Safety Concern of the Use of Probiotics and Transgenic Probiotics --- p.33 / Chapter 1.5 --- Aims --- p.35 / Chapter Chapter 2 --- Material and Methodology --- p.36 / Chapter 2.1 --- General Materials --- p.36 / Chapter 2.1.1 --- Chemicals --- p.36 / Chapter 2.1.2 --- Antibodies and Commercial Kits --- p.41 / Chapter 2.1.3 --- Bacteria --- p.43 / Chapter 2.1.4 --- Animals --- p.43 / Chapter 2.1.5 --- Cell Lines --- p.44 / Chapter 2.1.5.1 --- Mouse Colonic Epithelial Cells --- p.44 / Chapter 2.1.5.2 --- Mouse Macrophages --- p.44 / Chapter 2.2 --- Experimental Designs --- p.45 / Chapter 2.2.1 --- Construction of mCRAMP-Encoding Lactococcus lactis --- p.45 / Chapter 2.2.1.1 --- Enumeration of L. lactis --- p.45 / Chapter 2.2.1.2 --- Bacteriostatic Effect of mCRAMP on L. lactis --- p.46 / Chapter 2.2.1.3 --- Construction of mCRAMP-Encoding L. lactis --- p.46 / Chapter 2.2.1.4 --- Detection of mCRAMP Production by Western Immunoblotting --- p.50 / Chapter 2.2.2 --- In vivo Studies --- p.52 / Chapter 2.2.2.1 --- Survival of mCRAMP-Encoding L. lactis in Murine Colon --- p.52 / Chapter 2.2.2.2 --- Toxicity of mCRAMP-Encoding L. lactis --- p.53 / Chapter 2.2.2.3 --- Determination of mCRAMP Expression in Colon Tissue --- p.53 / Chapter 2.2.2.4 --- Induction of Colitis --- p.54 / Chapter 2.2.2.5 --- Probiotic and Sulfasalazine Treatment --- p.54 / Chapter 2.2.2.6 --- Clinical Symptoms --- p.56 / Chapter 2.2.2.7 --- Morphological Analysis --- p.56 / Chapter 2.2.2.7.1 --- Haematoxylin-Eosin (H&E) Staining --- p.56 / Chapter 2.2.2.7.2 --- Periodic Acid-Schiff (PAS) Staining --- p.58 / Chapter 2.2.2.8 --- Assessment of Apoptosis and Proliferation by Immunohistochemistry --- p.60 / Chapter 2.2.2.8.1 --- Determination of Cell Apoptosis by Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling --- p.60 / Chapter 2.2.2.8.2 --- Determination of Cell Proliferation by Proliferating Cell Nuclear Antigen (PCNA) Staining --- p.61 / Chapter 2.2.2.9 --- Determination of the Degree of Inflammation --- p.63 / Chapter 2.2.2.9.1 --- Colonic Myeloperoxidase (MPO) Activity --- p.63 / Chapter 2.2.2.9.2 --- Colonic Malondialdehyde (MDA) Level --- p.63 / Chapter 2.2.2.10 --- Fecal Microbiota Count --- p.64 / Chapter 2.2.2.11 --- mRNA Expression of Inflammatory Cytokines --- p.64 / Chapter 2.2.3 --- In vitro Studies --- p.66 / Chapter 2.2.3.1 --- Determination of Anti-inflammatory Effects of mCRAMP --- p.66 / Chapter 2.2.3.1.1 --- Cell Viability --- p.66 / Chapter 2.2.3.1.2 --- Determination of TNF-α Secretion under Stimulation of LTA and LPS --- p.66 / Chapter 2.2.3.1.3 --- Effects of mCRAMP on TNF-α Secretion Under Stimulation of LTA or LPS --- p.67 / Chapter 2.2.3.1.4 --- Effects of Pertussis Toxin (PTX) on the Inhibition of TNF-α Secretion by mCRAMP --- p.67 / Chapter 2.2.3.1.5 --- Nuclear Factor-κB (NF-κB) Luciferase Reporter Gene Assay in RAW 264.7 cells --- p.68 / Chapter 2.2.3.1.6 --- Determination of IκBα Expression and Phosphorylation by Western Immunoblotting --- p.69 / Chapter 2.2.3.1.7 --- Determination of Inducible Nitric Oxide Synthases (iNOS) and Cyclooxygenase-2 (COX-2) Expression by Western Immunoblotting --- p.70 / Chapter 2.2.3.2 --- Determination of Wound Healing Effects of mCRAMP --- p.72 / Chapter 2.2.3.2.1 --- Cell Viability --- p.72 / Chapter 2.2.3.2.2 --- Cell Migration --- p.74 / Chapter 2.2.3.2.3 --- Determination of Epidermal Growth Factor Receptor (EGFR), Extracellular Signal-Regulated Protein Kinase (ERK1/2) and p38 Expression and Phosphorylation by Western Immunoblotting --- p.76 / Chapter 2.3 --- Statistical Analysis --- p.76 / Chapter Chapter 3 --- Result --- p.77 / Chapter 3.1 --- Protective Effects of Cathelicidin-Encoding Lactococcus lactis in Murine Ulcerative Colitis --- p.77 / Chapter 3.1.1 --- Introduction --- p.77 / Chapter 3.1.2 --- Results --- p.79 / Chapter 3.1.2.1 --- Survival of mCRAMP-Encoding L. latis in Murine Colon --- p.79 / Chapter 3.1.2.2 --- Detection of mCRAMP-Encoded by L. lactis in vivo --- p.81 / Chapter 3.1.2.3 --- Toxicity of mCRAMP-Encoding L. lactis --- p.84 / Chapter 3.1.2.4 --- Clinical Symptoms --- p.86 / Chapter 3.1.2.5 --- Histology Evaluation --- p.89 / Chapter 3.1.2.6 --- Apoptosis --- p.93 / Chapter 3.1.2.7 --- Determination of mCRAMP Expression in Colon Tissue --- p.96 / Chapter 3.1.2.8 --- Determination of the Degree of Inflammation --- p.101 / Chapter 3.1.2.9 --- Faecal Microbiota Populations --- p.105 / Chapter 3.1.3 --- Discussion --- p.108 / Chapter 3.2 --- Therapeutic Effects of Cathelicidin-Encoding Lactococcus lactis in Murine Ulcerative Colitis --- p.113 / Chapter 3.2.1 --- Introduction --- p.113 / Chapter 3.2.2 --- Results --- p.115 / Chapter 3.2.2.1 --- Clinical Symptoms --- p.115 / Chapter 3.2.2.2 --- Histology Evaluation --- p.117 / Chapter 3.2.2.3 --- Cell Death and Proliferation in Colitis --- p.123 / Chapter 3.2.2.4 --- Determination of mCRAMP Expression in Colon Tissues --- p.127 / Chapter 3.2.2.5 --- Determination of the Degree of Inflammation --- p.130 / Chapter 3.2.2.6 --- Faecal Microbiota Populations --- p.133 / Chapter 3.2.3 --- Discussion --- p.135 / Chapter 3.3 --- Mechanistic Study of the Anti-inflammatory Effects of mCRAMP in Mouse Macrophages --- p.139 / Chapter 3.3.1 --- Introduction --- p.139 / Chapter 3.3.2 --- Results --- p.145 / Chapter 3.3.2.1 --- Viability of Macrophages --- p.145 / Chapter 3.3.2.2 --- Effects of LTA and LPS on Tumour Necrosis Factor-α (TNF-α) Release from Macrophages --- p.150 / Chapter 3.3.2.3 --- The Inhibition of TNF-α Secretion by mCRAMP --- p.153 / Chapter 3.3.2.4 --- Inhibition of TNF-α Secretion by mCRAMP Independent to GPCR Stimulation --- p.158 / Chapter 3.3.2.5 --- Activation of NF-κB Through Detection of Luciferase Activity --- p.163 / Chapter 3.3.2.6 --- Determination of the Expression and Phosphorylation of IκBα by Western Immunoblotting --- p.166 / Chapter 3.3.2.7 --- Determination of iNOS and COX-2 Expression by Western Immunoblotting --- p.169 / Chapter 3.3.2.8 --- The Suppression of iNOS and COX-2 Expression by mCRAMP was Independent to GPCR and P2X₇ Signalling --- p.183 / Chapter 3.3.3 --- Discussion --- p.188 / Chapter 3.4 --- Mechanistic Study on Wound Healing Effect of mCRAMP in Mouse Colon Epithelial Cells --- p.193 / Chapter 3.4.1 --- Introduction --- p.193 / Chapter 3.4.2 --- Results --- p.196 / Chapter 3.4.2.1 --- Cell Viability --- p.196 / Chapter 3.4.2.1.1 --- MTT Assay --- p.196 / Chapter 3.4.2.1.2 --- BrdU Incorporation --- p.200 / Chapter 3.4.2.2 --- Cell Migration --- p.202 / Chapter 3.4.2.3 --- Determination of Epidermal Growth Factor Receptor (EGFR), Extracellular Signal-Regulated Protein Kinase (ERK1/2) and p38 Expression and Phosphorylation by Western Immunoblotting --- p.210 / Chapter 3.4.3 --- Discussion --- p.215 / Chapter 4 Discussion and Future Perspectives --- p.219 / Publications --- p.224 / References --- p.227

A dextran sulphate (DS) model of ulcerative colitis was used to examine the effects of macronutrient restriction with (MR+PRO) and without (MR) probiotic supplementation (VSL#3RTM) on protein metabolism and growth. MR and MR+PRO decreased weight and chest circumference gain, but had no effect on linear growth of piglets. MR decreased the protein fractional synthesis rate (FSR) of liver, masseter, longissimus dorsi, colon, as well as plasma albumin, measured by stable isotope tracer L-[ring-2H 5]phenylalanine. MR+PRO increased the FSR of hepatic proteins by greater than 70% and increased both the FSR and absolute synthesis rate (ASR) of the total plasma protein pool, albumin and fibrinogen. Over 11 days, MR+PRO piglets showed a smaller decrease in plasma total protein concentrations than WN piglets, and maintained higher albumin levels than either WN or MR piglets. These findings highlight the importance of adequate nutrition during gastrointestinal inflammation and show that when food intake is decreased, probiotics stimulate liver protein synthesis and increase the acute phase protein response, similar to the well-nourished state.

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Both ulcerative colitis and Crohn's disease are chronic disorders of unknown aetiology. The purpose of this study was to map the incidence and current natural history of both diseases so that this knowledge might assist in the better management of patients. The study is of a cohort of 723 patients with inflammatory bowel disease diagnosed in Wolverhampton, Salisbury and Swindon between 1977-86 and followed up prospectively until 31st December 1993. The incidence, mortality, survival and short-term prognosis in relation to age, sex, length of history, severity and extent of disease were analysed. The long-term prognosis was examined in relation to the course, relapses, recurrence, surgical treatment, remote complications and cancer risk. No increase in the incidence of ulcerative colitis and Crohn's disease was found. The age distribution of both diseases showed a bimodal pattern with peaks at 20-39 and 50-69. The incidence of ulcerative colitis was highest in Wolverhampton as was the incidence of Crohn's disease in Swindon. It was similar between men and women. The Standardised Mortality Ratio (~MR) of ulcerative colitis and Crohn's disease was similar to the general population, except that it was significantly higher in Swindon. The excess death was not from ulcerative colitis. Ulcerative colitis: Rank correlation analysis showed length of history, extent and severity of disease were strongly associated with prognosis of first attack. There was also a strong inter-relationship between these factors. 80% of patients were in remission at any time but at 15 years 72% had experienced at least one relapse and 15% of patients had undergone colectomy. The severity and extent of disease at first attack was found to be predictive of colectomy. The prognosis was different in Wolverhampton. More patients underwent surgery and colectomy. When the influence of race was considered South Asian patients presented with more severe and extensive colitis and they were younger than white patients. The relapse and colectomy rate were greater in South Asians during follow up. The racial differences in incidence, course and prognosis of ulcerative colitis needs further investigations. The lower incidence in Swindon, less common extensive colitis both at presentation and during follow up, and lower colectomy rate might suggest a milder form of ulcerative colitis. Crohn's disease: Rank correlation analysis showed age, site of disease and length of history were not associated with short term prognosis. Almost 80% of patients were in remission at any time and 84% experienced at least one relapse in 14 years which required treatment. The relapse rate in Swindon was higher but were mostly treated medically.47% of patients had undergone some form of intestinal resection in 14 years, 33% required a second operation within 10 years. This study failed to show any relationship between the first recurrence and age at diagnosis, length of history or initial site of involvement. The prognosis was different in Wolverhampton; more patients underwent intestinal resection. In Wolverhampton there were significantly more patients with anal Crohn's and 38% of these patients who underwent surgery did so because of anal Crohn's. There was a improvement in the prognosis of VC and CD in the present study in comparison to the studies in the last few decades. The apparent improvement of outcome may result from changing criteria and speed of diagnosis, the introduction of new treatments and their rapid application. The need exists for new and improved treatment for those patients who do not currently respond adequately. Outcome measurements suggest that most, but not all patients, with ulcerative colitis and Crohn's disease lead a relatively normal social and working life. The results may be of value for reassessing the life insurance risk of patients with inflammatory bowel disease. The results suggest there is a need for further reallocation of resources for both research and health care delivery to these patients.

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