Inflammatory bowel disease (IBD) is a composite classification for a range of gastrointestinal disorders. The two most common forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). The chronicity of IBD has long-term consequences on the quality of life of affected individuals. However, therapies available today are applied with limited success. It is thought that interplay between environmental triggers and commensal bacteria can cause a dysregulated inflammatory response in genetically predisposed individuals, but the exact etiology is not understood. The objective of these studies was to identify the gene expression changes which associate with differential response to therapy and with recurrence of disease following surgery. The effects of genetic variation on gene expression were also evaluated. Tissue and blood samples were collected from eligible patients. Total RNA was extracted and measured on gene expression microarrays. The raw gene expression data were analyzed in a statistical framework against variables of interest in order to assess the significance of each association. Genes were evaluated individually as well as in biological networks. A few tens of genes were identified as differentially expressed in blood between UC patients who respond to intravenous corticosteroid therapy and those who do not. Some of these genes were also shown to have high predictive value. Differentially regulated genes were also found in UC patients who experience recurrence of disease after surgery, relative to those who remain disease-free. Specifically, gene networks responsible for the regulation of cellular transport were among the major players. A comparative analysis of genotype and gene expression in the human ileum indicated that the transcription of approximately 10% of genes is influenced by variations in the genome. Results from these studies have not only contributed to our understanding of disease mechanism, but could also have medical implications. With the advance of new and less costly transcriptome technologies on the clinical stage, panels of expression markers associated with therapy response and post-operative recurrence can be used as diagnostic and prognostic tools. Data on the relationship between genotype and gene expression are already shedding new light on the function of certain genetic IBD risk variants.

Inflammatory bowel disease (IBD) is a composite classification for a range of gastrointestinal disorders. The two most common forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). The chronicity of IBD has long-term consequences on the quality of life of affected individuals. However, therapies available today are applied with limited success. It is thought that interplay between environmental triggers and commensal bacteria can cause a dysregulated inflammatory response in genetically predisposed individuals, but the exact etiology is not understood. The objective of these studies was to identify the gene expression changes which associate with differential response to therapy and with recurrence of disease following surgery. The effects of genetic variation on gene expression were also evaluated. Tissue and blood samples were collected from eligible patients. Total RNA was extracted and measured on gene expression microarrays. The raw gene expression data were analyzed in a statistical framework against variables of interest in order to assess the significance of each association. Genes were evaluated individually as well as in biological networks. A few tens of genes were identified as differentially expressed in blood between UC patients who respond to intravenous corticosteroid therapy and those who do not. Some of these genes were also shown to have high predictive value. Differentially regulated genes were also found in UC patients who experience recurrence of disease after surgery, relative to those who remain disease-free. Specifically, gene networks responsible for the regulation of cellular transport were among the major players. A comparative analysis of genotype and gene expression in the human ileum indicated that the transcription of approximately 10% of genes is influenced by variations in the genome. Results from these studies have not only contributed to our understanding of disease mechanism, but could also have medical implications. With the advance of new and less costly transcriptome technologies on the clinical stage, panels of expression markers associated with therapy response and post-operative recurrence can be used as diagnostic and prognostic tools. Data on the relationship between genotype and gene expression are already shedding new light on the function of certain genetic IBD risk variants.

Biological markers (“biomarkers”) may have applications in inflammatory bowel disease (IBD), a chronic disease of the gastrointestinal tract. Clinicians are presented with several challenges when treating IBD. Instead of performing expensive and invasive endoscopic procedures - if even possible, as resources for these procedures can be limited - biomarkers could be used to diagnose, assess disease activity and prognosis, and guide medical therapy, particularly in situations where novel biologics are involved. At this time, the use of biomarkers is limited, since few have been useful in predicting disease severity, prognosis and therapeutic response in IBD. Previous research cohorts studying biomarkers are limited due to varying heterogeneity between subjects that confounds the results since patients have variable disease courses. The main aim of this work was to evaluate the utility of biomarkers in IBD. To do this, biomarkers were included into a composite score with other patient reported outcomes (PRO) to predict endoscopic disease activity. Next, we examined the role of biomarkers in newly diagnosed IBD. Lastly, fecal calprotectin (FC) was evaluated in healthy pregnant and IBD patients, establishing reference values and practicality in this clinical group. We also studied the relationship between biomarkers and environmental factors, such as fecal microbiota. We hypothesized biomarker concentration would be elevated with increased clinical and endoscopic measures, and predictive of response to medical therapy in newly diagnosed patients. Additionally, we theorized the inclusion of biomarkers into composite scores would outperform existing scoring models in predicting endoscopic severity. Furthermore, FC levels would be below the limit of detection in healthy pregnancy and elevated in IBD pregnancy. The inclusion of biomarkers into composite scoring models outperformed existing clinical scores. In newly diagnosed patients, modest relationships were found between biomarkers and clinical and endoscopic markers of disease. Lastly, the presence of FC was elevated in pregnant IBD and not significant in healthy pregnancy; thus, FC is useful in IBD and pregnancy. Our work confirmed the significance of biomarkers in several clinical areas of IBD, along with the issues presented in recruiting newly diagnosed patients in small research centres. Future work will incorporate biomarkers into medical triage and as an endpoint in nutritional interventions.

Chemistry of intermetallic bonded diamond is studied. The impact resistance and energies of intermetallic bonded diamond is compared to current poly crystalline diamond compacts. IBD’s are found to have high standards of hardness and have more impact energies absorbed. Intermetallic bonded diamond composite comprises of diamond particles dispersed in Tungsten carbide using Nickel aluminide (Ni3Al) as binder. In previous research conducted on IBD’s, diamonds are successfully dispersed in intermetallic alloy of nickel aluminide and processed at 1350°C such that diamond particles remain intact without forming graphite. Composites are formed by milling, pressing the intermetallic binder and diamond particles and sintering at high temperature conditions.

Inflammatory bowel disease (IBD) is a group of diseases in the gastrointestinal field that is becoming more commonly diagnosed among patients. IBD is usually characterized as a group of chronic diseases affecting the digestive tract that are caused by a multitude of factors including genetic, environmental, mucosal, and immune contributors. One of the subgroups of IBD is very early onset IBD (VEO-IBD), which is diagnosed in children under the age of 6. VEO-IBD is a rare yet unique case of IBD, which reports poor response to conventional adult-onset IBD treatments. Nutrition is an alternative treatment that can decrease inflammation and allow IBD patients to achieve remission. This proposed study explores whether formula-based diets, which have been strongly correlated with reduced IBD inflammation and symptoms, will impact VEO-IBD patients. A mouse model will be set up with one control group of healthy mice and two variable groups of VEO-IBD characteristic mice, with 60 mice in each group. The mice will be fed three formula-based dietary regiments including camel’s milk, Pediasure, and liquid vitamin D3 twice daily for 90 days. All three of these dietary treatments have been proven to decrease inflammation in adult-onset IBD patients. The inflammation and severity of symptoms will be monitored every two days through Western blotting protein levels of IL10 (a genetic marker for VEO-IBD) and physiological tests. If nutrition has a positive effect on the VEO-IBD induced mice, then a decrease in inflammation and VEO-IBD symptoms should be observed. This study is vital to future treatment plans by determining the influence of formula-based diets in alleviating symptoms of VEO-IBD patients.

In this thesis nanoparticles consisting of a calcium phosphate core encapsulated by poly(lactic-co-glycolic) acid and polyethylenimine were developed for the delivery of siRNA in vivo. The nanoparticles were efficiently endocytosed by different cell types in vitro without exhibiting cytotoxic characteristics. Without possessing endogenous immune response activating properties, the nanoparticles had a highly preferable composition for the delivery of siRNA and subsequent gene knockdown. The delivery of siRNA with nanoparticles was tested in two different murine disease models: DSS-induced colitis as model for human IBD and a TH1-induced lung inflammation as model for COPD. In IBD and COPD chemokines and cytokines are predominant players in the progression of the inflammatory response. The local interference of cytokine signalling mediated by siRNA-loaded nanoparticles might therefore be a promising new therapeutic approach. In both murine models, the aim was to deliver siRNA directed against inflammation related cytokines by nanoparticles for the local treatment of mucosal inflammation. The local administration of nanoparticles loaded with siRNA to mice suffering from intestinal or lung inflammation led to significantly decreased target gene expression on mRNA as well as protein level in biopsies from the target tissues. Furthermore, reduced cytokine levels were accompanied by diminished inflammatory pathologies and augmented clinical signs of sickness. The results of this thesis indicate that a specific and local modulation of inflammatory responses by nanoparticle-based siRNA delivery is feasible and demonstrates a major therapeutic potential.

Inflammatory bowel disease (IBD) is a chronic disease which steadily increases worldwide with the highest prevalence in Canada. Genetic susceptibility is considered to be an important factor in causing IBD. Organic cation transporters, SLC22A4 and SLC22A5 have been associated to IBD multiple times. Recently, SLC22A23, a novel gene that encodes for an organic cation membrane transporter protein has also been associated to IBD however; neither its gene structure nor its functions has been characterized. The aim of this study was to characterize the genomic structure of SLC22A23 gene using bioinformatics analysis, determine the tissue expression, characterize the location of the protein and perform functional studies using Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry. We have identified the chromosomal location, the gene neighborhood and the genomic structure of human SLC22A23.The result of this study indicates that SLC22A23 gene is a membrane transporter and it is abundantly expressed in the intestine. / October 2015

Reduced bone mass is a common complication of human inflammatory bowel disease, however, the mechanisms that contribute to osteopenia are not completely understood. Cytokines are up regulated in IBD patients and have been shown to have detrimental effects on osteoblasts. PHEX is expressed predominantly in osteoblasts; disruption of the PHEX gene results in defective bone mineralization and renal phosphate wasting. We hypothesize that PHEX gene expression as well as overall Pi homeostasis are altered in individuals with IBD and therefore, may contribute to alterations in bone mineral density observed in individuals with IBD. In vivo studies: 6-7 week old Balb/C mice were intrarectally instilled with TNBS or 50% ethanol. Animals were treated with or without neutralizing anti-TNFα antibody, dietary curcumin, or systemically with recombinant TNFα. RNA was prepared from bone and gene expression was analyzed by PCR. Kidney and small bowel were harvested from control and TNBS treated animals and embedded in paraffin for immunohistochemical analysis. In vitro studies: Cells were treated with IFNγ, IL1-β, IL-6, or TNFα and RNA was collected for real-time PCR analysis. UMR-106 cells were transfected with Phex promoter constructs. PHEX is down-regulated in mice with chemically induced colitis and in the mice injected with TNFα, this decrease was attenuated by curcumin and TNFα antibody. TNFα decreased endogenous levels of PHEX mRNA, protein, and inhibited spontateous mineralization in UMR-106 cells. This regulation occured at a transcriptional level and it appears that the proximal poly-A region of the PHEX promoter played a significant role in downregulation of Phex expression. Phosphatonin expression was not altered in TNFα-treated UMR-106 osteoblasts. Cytokines were unable to alter phosphatonin mRNA expression in UMR-106 cells and no changes in phosphatonin expression were observed in vivo. Intestinal NaPi-IIb mRNA expression decreased in TNBS-treated animals although immunohistochemical analysis did not reveal any changes in cellular localization of NaPi-IIb protein. Renal NaPi-IIa mRNA did not change in TNBS-treated animals however, immunohistochemical analysis revealed internalization of NaPi-IIa from the apical membrane. In conclusion, decreased phosphate absorption in the kidney along with altered Phex gene expression may contribute to decreased bone mineral density observed in IBD patients.

OBJECTIVES: Inflammatory bowel disease (IBD) is a chronic condition with increasing pediatric incidence. Transition from pediatric to adult care is associated both with disruption of links with familiar caregivers and differences in health care delivery. This study explored the impact of the transfer from pediatric to adult care on health services utilization for pediatric-onset IBD patients. APPROACH: A population-based retrospective cohort study identified all children diagnosed with IBD 1994-2008 and treated by pediatric gastroenterologists from within Ontario health administrative data. Self-controlled case series analyses compared health services utilization in the 2 years before and after transfer to adult gastroenterologists, with a 6-month washout period at transfer. Outcome evaluated included IBD related/specific hospitalization, emergency department (ED) utilization, outpatient visits, and laboratory utilization. Relative incidence (RI) in the post-transfer was compared to pre-transfer periods using Poisson regression analysis controlling for transfer starting age. Analyses were stratified by IBD types: Crohn’s disease (CD) and ulcerative colitis (UC). RESULTS: 536 patients were included in the study (388 CD, 148 UC). ED utilization rate was higher after transfer for both CD (RI 2.12, 95% CI 1.53-2.93) and UC (RI 2.34, 95% CI 1.09-5.03). Other increases in health care provision included outpatients visits (CD: RI 1.56, 95% CI 1.42-1.72; UC: RI 1.48, 95% CI 1.24-1.76), and laboratory investigations (CD: RI 1.43, 95% CI 1.26-1.63; UC: 1.38, 95% CI 1.13-1.68). There was no statistically significant change in hospitalization (CD: RI 0.70, 95% CI 0.42-1.18; UC: RI 2.41, 95% CI 0.62-9.40). Sensitivity analysis revealed similar results when only the first year post-transfer period was assessed. CONCLUSIONS: In the largest study to date examining the transfer from pediatric to adult IBD care, health services utilization increased significantly in the two years after transfer for measures of access to health care providers. Understanding causation and rigorously evaluated transition programs may help to better manage the resources and meet the needs of pediatric-onset IBD patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19571号 / 医博第4078号 / 新制||医||1013(附属図書館) / 32607 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 武藤 学, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM