Recent advances have increased the understanding that dysbiosis of the gut microbiome may be a significant contributor to the pathophysiology of ulcerative colitis. Because of this, the use of fecal microbiota transplants (FMT) has become more popular as a potential supplemental treatment option for patients suffering from this disease. Research has shown a possible benefit of FMT in conjunction with varying conventional therapies for patients with mild to moderate disease severity. However, there are scarce publications that have investigated the benefit of FMT in conjunction with a single conventional therapy for patients with moderate to severe disease, specifically. The proposed study is a multicenter, double blind, randomized controlled study of FMT, mercaptopurine (6-MP), and prednisone vs 6-MP and prednisone alone in patients with moderate to severe ulcerative colitis. The study subjects will have a baseline evaluation and the treatment trial will last 8 weeks with follow up throughout the study. Investigators will analyze the primary outcome of clinical remission and secondary outcomes of improvement of fecal calprotectin levels, Inflammatory Bowel Disease Questionnaire (IBDQ) score, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in the treatment vs control groups. The data from this study will help to identify if FMT would be an additional safe, efficacious treatment modality to the current medical management of ulcerative colitis.

Background: Reliable biomarkers are needed to evade the risk of injury, invasiveness and discomfort of endoscopies, which are required for inflammatory bowel disease (IBD) diagnosis and extent of disease assessment in ulcerative colitis (UC) patients. The need for biomarkers is accentuated in children, wherein the most frequently used IBD biomarker yields low specificity. Proteomics of clinical samples or their enriched components is a means to evaluate and identify alterations in proteins reflective of disease, with the potential for use as biomarkers and for providing insight on disease pathogenesis. Methods: Proteins were isolated from the intestinal mucosal-luminal interface (MLI), collected from the ascending and descending colon of pediatric treatment-naive patients. The intestinal MLI proteomes of 42 IBD and 18 control patients were analyzed by high resolution mass spectrometry (HRMS). Multivariate analysis and receiver operating characteristics curves were performed to develop protein biomarker panels to discriminate IBD from control, and for UC extent of disease. ELISAs were used to assess a subset of biomarker candidates in stool samples from an independent pediatric cohort (n=24). Extracellular vesicles (EVs) were isolated by ultracentrifugation from the intestinal MLI of 11 IBD and seven control patients, and analyzed by electron microscopy, nanoparticle tracking analysis and HRMS. Results: A biomarker panel of four proteins classified patients as either controls or active IBD with 97.5% accuracy. A second biomarker panel correctly classified 100% of UC patients as presenting with pancolitis or non-pancolitis. The differential protein expression of two biomarker candidates (catalase and leukotriene A-4 hydrolase) identified from the intestinal MLI was comparable in stool samples. Comparison of EV proteomes isolated from IBD patients and controls identified differential expression of processes related to host defense and immunity. Conclusions: Proteomic analysis of clinical samples identified differentially expressed proteins that can classify IBD patients from non-IBD controls and distinguish UC patients with pancolitis from those without pancolitis; proteins identified in intestinal aspirates displayed consistent differential expression in stool. Furthermore enrichment of EVs from the intestinal MLI indicates that these may contribute to the dysregulated host response against the intestinal microbiota which is observed in IBD.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17816号 / 医博第3814号 / 新制||医||999(附属図書館) / 30631 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 一山 智, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

BACKGROUND: Early identification of pediatric patients with Inflammatory Bowel Disease (IBD), including ulcerative colitis and Crohn disease, is important to help clinicians design optimal treatment regimens. Existing endoscopic techniques are effective in identifying disease activity. However, these methods are invasive, expensive, and less amenable to serial measurement. Recent studies have identified potential serologic and fecal biomarkers that may have the potential to provide clinicians with a more objective evaluation of disease activity. In the case of ulcerative colitis (UC), in which disease is confined to the large intestine, the information provided by fecal biomarkers is likely to be more specific than that provided by serologic biomarkers. Fecal lactoferrin (FLA) is one such biomarker that has shown to be useful not only in identifying levels of colonic inflammation, but also for use as a predictor of disease relapse and treatment efficacy. Measurement of fecal lactoferrin, in conjunction with information provided by other diagnostic modalities could expedite patient assessment and treatment. Additionally, it has been suggested that fecal lactoferrin levels may also provide prognostic information about response to treatment and disease outcome in pediatric patients with UC. The goal of this study is to explore the relationship between changes in FLA levels and response to medical therapy in hospitalized pediatric patients with UC. METHODS: Serial stool samples were collected daily from 10 patients admitted for management of severe active UC. Of these 10 patients, 3 responded favorably to standard treatment with intravenous corticosteroid therapy and were discharged to complete a course of oral steroids. 7 were unresponsive to steroid therapy and went on to require rescue (more intensive) medical therapy. Changes in FLA were correlated with steroid response and medical disposition at the time of discharge. RESULTS: A t-test was performed to determine the significance of the differences in percent change in FLA levels between patients discharged on steroids and patients discharged on rescue therapy. Patients discharged on steroids demonstrated a net decrease in FLA levels over the course of the first three days of steroid treatment while patients ultimately requiring rescue medical therapy demonstrated a net increase in FLA levels (mean values = -64.4% and +203.8%, respectively). A difference was found between the averages; however, this value did not reach statistical significance when analyzed with a t-test (p = 0.18). CONCLUSIONS: This study suggests that quantitative FLA levels may prove useful in predicting clinical course and discharge outcome in pediatric patients with ulcerative colitis. Future research in this field should seek larger sample sizes, increased longitudinal sample collection, and the potential for a composite assessment that will yield additional objective measures of disease activity.

The collection of microbes that inhabits the human gastrointestinal tract is known as intestinal microbiota, and an enormous body of work has shown that their activities contribute to health and disease. Ulcerative colitis (UC), which is a type of inflammatory bowel disease, is considered to arise due to a disruption in the balance between the immune system and microbiota. However, there is little consensus on the mechanism of action and microbes involved in the disease manifestation. In this work, I applied culture-enriched metagenomics (CEMG) to characterize the dynamics of gut microbiota in healthy individuals and UC patients. I showed that CEMG provides a higher resolution to study these microbial communities, and we used this approach to understand microbial colonization after fecal microbiota transplantation (FMT) therapy in UC patient. I showed that sequencing approaches alone did not reveal consistent engraftment across FMT responders. Using CEMG and a collection of bacterial whole-genome sequences, I showed patient-specific microbial strain transfer and a signature of commonly engrafted genes only in patients who responded to FMT. In this work, I also investigated the dynamics of a highly abundant bacteriophage, crAssphage, in an FMT donor and implemented a new method to detect bacteriophage engraftment post-FMT using SNP analysis. Finally, it has been suggested that antibiotic treatment before FMT may increase the efficacy of FMT. However, in this work, I show that while antibiotics alter the microbiome, there was no difference in the composition of the microbiome of antibiotic vs placebo group post-FMT. This is consistent with the randomized controlled trial results that shows pretreatment with antibiotics does not improve FMT outcome. Together, this work demonstrate the importance of in-depth microbiome analysis applied to culture-dependent and -independent sequencing to characterize microbial changes post-FMT. / Dissertation / Doctor of Philosophy (PhD) / Many bacteria reside in the human gut, and they are essential in our health and in disease. It is evident that these bacteria are associated with inflammatory bowel disease, but we do not yet know how and what bacteria are involved in this disease. In this work, I describe a method to study these bacteria from stool that relies on growing them and investigating their DNA. I showed that our approach helped us recover a greater diversity of these bacteria and their genetic content in healthy individuals and patients with inflammatory bowel disease compared to methods that use only DNA based approaches. Using this method, we could better understand why some patients responded to a treatment consisting of transferring stool content from healthy donor to patient. I also investigated a group of viruses that infect bacteria and implemented a new computational method based on DNA sequencing to test whether these viruses transfer to the patient after receiving the fecal therapy. We also found that antibiotic treatment before fecal therapy in patients with inflammatory bowel disease does not improve the patient’s recovery.

It was the purpose of this thesis to establish the functional level of the thyroid gland in patients with chronic ulcerative colitis.

The inflammatory bowel diseases (IBDs) are a group of chronic conditions affecting the gastrointestinal tract, often presenting with non-specific clinical features such as abdominal pain, weight loss and diarrhoea. Approximately 25% of patients are diagnosed with IBD in childhood. For epidemiological studies, previously collected (1990-1995) and original (2003-2008) Scottish incidence data were used to determine national trends in newly diagnosed paediatric IBD (PIBD). A smaller, geographically defined, prospective 14-year cohort (1997- 2011) in South-East Scotland (SES) was used to assess regional trends in incidence, point prevalence, disease extent, medication use and PIBD surgery rates in 326 children. For the detailed analysis of the role of ICOSLG and CRP in Scottish children with PIBD, haplotype-tagging of both genes in 448 children (and their parents) registered on the Paediatric Inflammatory bowel disease Cohort and Treatment Study (PICTS) database was performed. Further clinical information from this database and previously gathered adult mRNA microarray data were also used to inform the analysis. For the faecal calprotectin (FC) case-control study, all PIBD patients diagnosed in SES between 01.01.05 and 31.12.10 (aged 1- 17yrs) with a FC performed during initial workup were identified; controls were matched non- IBD patients who had similarly undergone endoscopy with a referral FC level available. The systematic review and meta-analysis of FC case-control studies was performed with keywords relating to IBD and calprotectin in electronic resources from 1946 to May 2012. Inclusion criteria were studies that reported FC levels prior to the endoscopic investigation of IBD in children less than 18 years old. Laboratory work used newly derived HEK293 and HCT116 cell lines stably expressing wild-type NOD2 and the CD-associated NOD2 frameshift mutant, as well as utilising previously derived HEK293 and HCT116 cells stably expressing green fluorescent-labelled protein LC3 during the assessment of autophagy. Western blot, immunofluorescent microscopy and flow cytometry were used for analysis. There was a significant rise in PIBD incidence in Scotland since the early 1990s, with 260 new cases between 1990-1995 (4.45/100,000/year) and 436 in the 2003-2008 epoch (7.82/100,000/year) (p<0.001). A five-fold increase in Crohn's disease (CD) in the last 40 years was also demonstrated. SES was shown to have the highest recorded PIBD incidence rate in the UK for the six-year epoch from 2006-2011 (9.50/100,000/year) with a significant rise in ulcerative colitis (UC) to 2.67/100,000/year (p=0.010). Point prevalence rates for PIBD in SES had also risen significantly to 41.2/100,000 between the 2000-2005 and 2006-2011 epochs (p=0.016). With a follow up of 1577 patient years, the severe phenotype in children with PIBD was confirmed; 34% of children with CD presented with pan-enteric disease (44% at follow up), and 76% of children with UC had pancolonic disease at diagnosis (81% at follow up). 26% of patients required methotrexate and 18% were exposed to infliximab/adalimumab, with the time to first exposure of both significantly lower in children diagnosed between 2006-2011 (p=0.001 and p<0.001 respectively). A total of 70% of children were exposed to azathioprine and 20% underwent IBD-related surgery. Using a haplotype-tagging approach and transmission disequilibrium testing (TDT) in 230 PIBD case-parent trios there was significant overtransmission of the rs8126734-A single nucleotide polymorphism (SNP) in ICOSLG following correction (p=0.0467). In the CD TDT analysis the same SNP was overtransmitted (p=0.0084). The strongest susceptibility signal was evident across the two marker haplotype rs762421-A / rs8126734-G (p=0.0072), suggesting that the 3-prime untranslated region in ICOSLG may be targeted for deep sequencing. mRNA microarray data from adult patients showed downregulation of ICOSLG expression in the ascending colon (p=0.023) and upregulation in the descending colon (p=0.0351) in uninflamed biopsies from CD patients and non-IBD controls; no difference in gene expression was shown in UC patients. Using a similar approach, the A allele of two SNPs tagging CRP showed significant over-transmission to affected IBD patients after correction (rs1417938, p=0.006; rs1130864, p=0.015). The six-marker haplotype (ACACAC) showed significant distortion of transmission to affected individuals (p=8x10-4). CD and UC patients demonstrated differences in rs1205 genotype (p=0.0085) and CRP haplotype (p=0.0024), with the influence of the rs1205 SNP on response to anti-tumour necrosis factor-alpha therapy also shown (p=0.021). During the FC case-control study significantly elevated FC levels at diagnosis were demonstrated compared to controls (1265 μg/g vs 65 μg/g; p<0.001). FC also outperformed commonly used blood parameters (e.g. CRP, ESR, platelets), with an area under the curve of 0.93 (95% CI 0.89-0.97) and good sensitivity (0.93 [95% CI 0.86-0.98]) and specificity (0.74 [95% CI 0.64-0.82]) when values above 200μg/g were used. FC levels were not influenced by disease location in CD or UC. The systematic review and meta-analysis highlighted the often poor methodological quality of previous studies and concluded that across all studies FC had a pooled sensitivity of 0.98 (95% CI 0.95-1.00) and pooled specificity of 0.68 (95% CI 0.50-0.86) for PIBD at diagnosis. Characterisation of cells stably-expressing wild-type NOD2 or the CD-associated NOD2 frameshift mutation demonstrated increased cell proliferation compared to empty vector, and an accentuated apoptotic response to serum starvation. The NOD2 frameshift protein had a shorter half-life (at 11 hours) than the wild-type protein, with degradation of the NOD2 protein shown to be mediated through a proteasome-dependent pathway, possibly through lysine residues on the CARD domain. Following the establishment of a robust method of assessing autophagy in a cell culture system, experimental work showed that muramyl dipeptide-induced autophagy is unlikely to signal through the mammalian target of rapamycin, with the intermediate filament vimentin shown to be intimately involved in this pathway; the vimentin gene (Vim) was also shown to be a candidate susceptibility gene for CD. Using a panel of PIBD drugs azathioprine was shown to induce autophagy in a dose-dependent manner through an mTOR-dependent, ERK-independent pathway. It can be seen that with the increasing incidence and prevalence of PIBD in Scotland that a greater understanding of epidemiological trends, the role of genetic susceptibility, the optimal use of biomarkers and translational functional biology are all needed to understand further the aetiopathogenesis of PIBD. This future work will undoubtedly help to inform service design and the clinical care pathways utilised to provide the best care for children in addition to targeting pathways for potential drug development, with these measures helping to prepare for the increasing disease burden generated by PIBD.

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

Background The conventional ileostomy can be avoided. Many attempts have been performed. The first successful solution was the continent ileostomy- Kock pouch. The high rate of complications and revisions some experienced forced surgeon to try to restore the continence by the mechanism of the anus involving an ileal pouch. Both procedures afterwards documented excellent functional outcome, but the complication rates were not negligible and the long-term failure rate were increasing. Different surgical refinements were done and the risk factors for complications and failures were investigated as experience and materials increased. Restoring of the integrity of anal function and the succsess of the ileal pouch-anal anastomosis shadowed the practise of the forerunner: the continent ileostomy reservoir. This latter procedure was more demanding and seemed in the first year of ileal pouchanal anastomosis era to have significant more complications and revisional surgery. The worldwide adoption of the pelvic pouch decreased the need for the continent ileostomy and a vicious circle evolved. Today only few centres perform the procedure. Patients who are not suitable for ileal anal-pouch anastomosis are seldom offered the possibility of having a continent ileostomy. Aims The aims of the study was to investigate surgical load, complications and long-term functional outcome and to define factors which affect these subjects in patients operated with ileal pouch-anal anastomosis, continent ileostomy or both in one single surgical department during the same period and without any institutional learning curve, and furthermore, to compare and contrast the two options. Material and methods From 1984 to 2005(7) 304 (315) patients were operated with IPAA at St. Olavs Hospital (earlier: Regional Hospital of Trondheim). From 1983 to 2002(7) 50 (65) patients had a continent ileostomy constructed. This was an observational study in the scope of surveillance and quality assurance. All patients were offered a planed regularly annual outpatient clinic follow up programme including a prospective standardised interview on clinical outcome. This was a supplement to clinical investigation with endoscopy and consecutive documentation of complications and other factors affecting the patients’ health. Data were recorded in the medical chart. In this system, all patients had recorded dataset. However, the intervals between data recordings differ and the intervals increased by time. All inpatients data were included. Standard descriptive statistical analysis and simple associations were undertaken. Handling longitudinal data with limited cases, varying time intervals was done in a Times Series Cross Sectional data model, analysed, and adjusted for several factors affecting functional outcome. Multivariable analysis was done. Results The estimated failure rate at 20 years was 11.4% for ileal pouch-anal anastomosis and 11.6% for continent ileostomy. Salvage procedures rates were 31% vs. 38%, respectively (p=0.06). The salvage procedures in IPAA included local procedures and redoes with laparotomy. Salvage procedures in CI were related to the function of the nipple valve, mainly nipple valve sliding and less frequent stenosis or fistulas. Complications rates were high. In pelvic pouch surgery, half of the patients would need re-operations in 20 years. Ten percentages had early anastomotic separation without septic complications. Four percentages had early pelvic septic complications. Fistulas and sepsis at the anastomotic site were the main severe complications, often leading to pouch failure. Closing of the loop ileostomy was accompanied with complications in six percentages. In the patients (48) who did not have a covering stoma the overall complications rate did not differ from those with a loop ileostomy, although nine needed a secondary stoma. Covering stoma seems to postpone anastomotic complications. Handsewn anastomosis had more strictures, but otherwise the complications rates were similar to stapled anastomosis. Patients having the diagnosis changed to Crohn`s diseases had more complications and higher failure rate. Early anastomotic complications were associated with long-term complications. In patients with continent ileostomy the nipple valve sliding is the main cause of revision. One third needed revision once or several times. At 20 years follow-up, half of the patients would need surgery due to complications. Although many patients with CI need several revisions, all patients were continent at the last follow up with a stable intubation frequency of 3 – 5 per 24 hour. The failure of the pelvic pouch is the end of severe complications. Two third of the failures had the pouch excision or permanent ileostomy with the pouch in situ. One third underwent a conversion to CI, with equal surgical and functional outcome as other patients with CI. In IPAA, bowel movements at day were between 5-6 at day and 0-1 at night. The rates of more or less frequent incontinence were about 10%, and 41% and 55% had reported soling at day and night respectively. The long-term functional outcome did not deteriorate with time: ie. observational time, as an independent factor did not influence outcome. Factors influencing the outcome were found but the impact of gender, age, protective stoma, hand-sewn anastomosis and early complications were negligible. Pouchitis did significantly influence functional outcome negatively, but did not create deterioration over time. Estimated pouchitis rate in IPAA was 43% for more than 20 years. The onset of the first pouchitis appears mostly in the 5-6 first years after surgery. The crude rate was 35% and 6% of the patients had chronic pouchitis. Severe/chronic pouchitis was associated with primary sclerosing cholangitis, but not with pyoderma gangrenousum or diagnosed joint affections. Idiopathic pouchitis were absent among patients with familial adenomatous polyposis. In continent ileostomy the rate of pouchitis was 26%. Conclusion The complications in both the pelvic pouch surgery and the surgery of continent ileostomy are considerable. Although not similar the surgical load are in the same order of magnitude. For the continent ileostomy revisional surgery are to be expected. The failure rate of both procedures are high and in long-term similar. The long-term functional outcome are however stabile and excellent. The failed pelvic pouch can be converted to a continent ileostomy in selected and motivated patients. The entity of pouchitis is conflicting and has to be divided into several different entities both on clinical, constitutional and other differentiating features. Patients with PSC should be informed of a possible higher risk of severe and chronic pouchitis after IPAA.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 112-126).