Thesis (Ph.D.) - University of Glasgow, 2009. / Ph.D. thesis submitted to the Division of Developmental Medicine, Faculty of Medicine, University of Glasgow, 2009. Includes bibliographical references. Print version also available.

Inflammatorisk tarmsjukdom blir allt vanligare. Sjukdomen är kronisk och kommer följa patienten för resten av livet. Sjuksköterskan behöver kunskap om patientens upplevelser av att leva med inflammatorisk tarmsjukdom för att kunna ge stöd och god omvårdnad. Syftet med litteraturstudien var att belysa patientens upplevelser av att leva med inflammatorisk tarmsjukdom. Metoden för litteraturstudien utformades efter niostegsmodellen av Polit och Beck (2012). En systematisk litteratursökning gjordes i databaserna Cinahl, PsycInfo och PubMed. En urvalsprocess i tre steg gjordes, där artiklarna bearbetades och kvalitetsgranskades. Elva artiklar inkluderades till resultatet, varav åtta kvalitativa studier, en kvantitativ studie och två kvalitativ-kvantitativ mixade metoder. Resultatet framställdes i sju kategorier som belyser patientens upplevelser av att leva med inflammatorisk tarmsjukdom. Kategorierna presenterar maktlöshet och frustration, social isolering, oro och rädsla, upplevelse av smärta, upplevelse av fatigue, att känna skuld och att känna kontroll över sin sjukdom. Litteraturstudiens slutsats beskriver att inflammatorisk tarmsjukdom bidrar till begränsningar i vardagen. Sjuksköterskan behöver kunskap om patientens upplevelse av att leva med inflammatorisk tarmsjukdom för att kunna ge stöd, och på så sätt bidra till att patienten lär sig leva med sin sjukdom.

The aetiology of IBD is unknown, but available evidence suggests that an aberrant immune response towards the commensal microbial flora is responsible for intestinal inflammation in genetically susceptible individuals. Studies from animal models of intestinal inflammation have greatly advanced our understanding of the immunological basis of IBD. However, translation of results from animal research into human studies is essential in order to improve treatment options and patient quality of life. In this thesis we present the successful introduction of translational studies on human tissue in our laboratory. In particular, we evaluated the role of the IL23/IL17 pathway in the human immune response and its role in IBD. IL23-driven inflammation has been primarily linked to its activity on Th-17 cells; however, work from our laboratory has identified a novel population of IL23-responsive ILC, which are responsible for innate colitis in mice. Here we have analyzed the role of IL23-responsive innate cells in IBD. Our results show increased expression of Th-17 signature genes amongst intestinal CD3- cells in patients with IBD. Furthermore, we observed a marked and selective increase in IL17 producing CD56- ILC in the inflamed intestine of patients with CD. ILC may contribute to intestinal inflammation through secretion of cytokines, such as IL17A and IL17F, and recruitment of other inflammatory cells, representing a novel tissue-specific target for the treatment of IBD. In addition, we present here our preliminary data on the characterization of human intestinal and systemic DC populations. In particular, we aimed to evaluate if in the context of the intestinal microenvironment DC develop specific regulatory features, as observed in murine CD103+ DC. We show that human intestinal DC populations exhibit specific regulatory properties, such as expression of genes associated with TGF-β and RA activity. Furthermore, CD103+ DC are present in the human gut and are characterized by tolerogenic markers. Remarkably, patients with IBD have reduced frequencies of intestinal CD103+ DC, which display a more pro-inflammatory phenotype. Alteration in DC subset composition and functional activity may result in a distort balance between immune effector and regulatory responses, promoting the development of intestinal inflammation.

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.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Bilirubin influence on the progression of inflammatory bowel disease Diploma thesis Anna Patková Supervisor: doc. PharmDr. Petr Nachtigal, Ph.D. Background: Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gut caused by an interaction of genetic and environmental factors. It is thought that tissue damage is also partly caused by an oxidative stress. Heme oxygenase-1 and bilirubin are strong antioxidants and both of them provide an anti-inflammatory effect in various tissues. The aim of this diploma thesis was to detect changes of expression of HO-I in the large intestine of normobilirubinemic and hyperbilirubinemic rats after the induction of acute or chronic experimental colitis. Methods: We used Gunn rats with hereditary defect of UDP-glucuronyltransferase, which causes hyperbilirubinemia. The control group of animals was made up of heterozygous littermates of the Gunn rats, which have normal serum bilirubin levels. All animals were treated by dextran sulfate sodium in order to induce an experimental colitis. Rats were divided into two groups. Each of them contained hyperbilirubinemic and normobilirubinemic...

During cell activation the tightly compacted DNA is made available to DNA-binding proteins allowing the induction of gene transcription. In the resting cell, DNA is packaged into chromatin whose fundamental subunit is the nucleosome, composed of an octamer of four core histones (H) 3, 4, 2A and 2B. During the induction of gene transcription, modification of histones, by acetylation, methylation etc., results in unwinding of the DNA, permitting access of large DNAbinding proteins, such as RNA polymerase II, and subsequent induction of gene transcription. This investigation initially examined the effects of pro-inflammatory stimuli LPS and TNF-a on the production of IL-8 in a macrophage cell line (U937 cells) and in two T-cell lines (Jurkat and HUT-78 cells) as a marker of NF-KB-directed inflammatory gene expression. The ability of dexamethasone (Dex) and triamcinolone acetonide (TA) (synthetic glucocorticoid agonists) to suppress expression of the inflammatory cytokine IL-8 and to regulate histone acetylation was also investigated in these cells. LPS and TNF-a caused an increase in IL-8 expression, which was further enhanced by the histone deacetylases inhibitor trichostatin A (TSA), suggesting a role for histone acetylation in IL-8 production in these cells. Dex and TA, repressed LPS- and TNF-a -induced IL-8 expression in all three cell lines. This effect of both Dex and TA was attenuated by TSA in all cell lines studied, where the effect of TSA was greater in TA stimulated cells. Stimulation of all cell lines with LPS and TNF-a induced acetylation of H4 lysine residues (K5, 8, 12 and 16), the highest elevation of which was for K8 and K12. Also demonstrate is a K5 and K16 specificity of acetylation by glucocorticoids, apparent in all cell lines studied. Dex and, to a greater extent, TA suppressed LPS- and TNFa-induced K8 and K12 acetylation. TSA attenuated the inhibitory effect of the glucocorticoids for all three cell lines. An inCrease in HDAC activity with GCs was observed and ChiP assay showed these events occur on the native IL-8 promoter via histone acetylation. Further studies investigated whether there were any links between histone acetylation and the regulation of apoptosis. It was showed that TSA induced apoptosis in cells previously stimulated with the inducer of oxidative stress hydrogen peroxide (H20 2). Studies into the activation of caspase 3 in LPS- and TNF-a stimulated cells revealed that the combinatory effect of Dex or TA with TSA Significantly enhanced expression of the marker in all three cell lines. In resting cells, Dex, and TA, in the presence of TSA downregulated caspase 3 expression. These findings support the notion that glucocorticoid actions on apoptosis is mediated, at least in part, through an action on histone acetylation. Finally, histone acetylation was investigated in vivo in two rat models of inflammation and in human subjects with inflammatory bowel disease (IBD). The results showed an increase in histone H4 acetylation lysine specificity of acetylation on K8 and K12 in inflamed tissue and Peyer's patches in animal models and in IBD patients. Whereas H3 acetylation was not elevated to the same extent in tissue and was restricted to the mantle zone of Peyer's patches. In general, the present studies on histone acetylation and inflammation (in animal models and IBD patients) underlined the possibility of a general mechanism linking activation of the transcription factor NFKB with histone acetylation. The ultimate objective of this work is to aid in the understanding of the mechanisms of how deregulation of chromosome structure leads to progression of the disease state. This knowledge may aid in the development of new therapeutic approaches or improved glucocorticoids.

The two options for treatment of the inflammatory bowel disease Crohn’s Disease are surgery and pharmacotherapy. Pharmacotherapy with the goal of inducing and maintaining remission is the preferred treatment route, but the current medications are not entirely effective in achieving these goals. Approximately half of Crohn’s Disease patients will be required to have surgical bowel resection within 20 years of diagnosis, and many patients are at higher risk of adverse events such as cancer, either directly as a result of Crohn’s Disease or due to side-effects of the drugs used to treat the condition. Medical management of the disease is very complicated and there is a relative lack of uniformity in treatment. Different drugs used either in monotherapy, sequential therapy, or combination therapy produce differing levels of efficacy and different outcomes. This analysis provides an overview of the four major classes of drugs used in the treatment of Crohn’s Disease and a discussion of the overall efficacy of the different methods of treatment. While more studies need to be conducted into the differing outcomes of monotherapy, sequential therapy, and combination therapy, it appears as though any treatment involving the use of biologics such as tumor necrosis factor alpha (TNF-alpha) inhibitors results in improved outcomes relative to treatment styles that lack the use of biologics.

BACKGROUND: Patients suffering from inflammatory bowel disease (IBD) are at increased risk of vitamin D deficiency. Daily or weekly vitamin D supplementation has not proven to be effective in improving vitamin D status, and it is thought that this failure has been primarily due to a lack of compliance. Circulating vitamin D is crucial to bone growth and development in children and adolescents. However, more recent data has demonstrated that vitamin D also plays a significant role in the maintenance and regulation of the immune system. OBJECTIVES: The primary aim of this study is to investigate the safety and efficacy of administering high dose oral vitamin D therapy in pediatric patients with IBD. We chose to study patients receiving Remicade, an immunosuppressive monoclonal antibody therapy administered intravenously, as the need for scheduled hospital-based infusions provides a unique opportunity to ensure compliance in our study population. METHODS: We identified consecutive pediatric patients with IBD with a recent 25-hydroxyvitamin D (25OHD) level < 30ng/mL, maintained on Remicade, and with no history of kidney or liver disease for inclusion in the study from November 2017 and November 2018. Enrolled patients received one-year of open-label therapy. Vitamin D treatment doses were assigned by Remicade interval and patients received either 50,000 international units (IU) (every 4-5 weeks) or 100,000 IU (every 6-8 weeks) vitamin D3 orally at the time of their Remicade infusions. In addition to vitamin D levels, spot urine calcium to creatinine ratios, serum calcium, phosphorus, and blood urea nitrogen (BUN) levels, quality of life metrics, and surveys pertaining to dietary vitamin D intake and ultraviolet B (UVB) radiation exposure were collected throughout the study period. RESULTS: Baseline vitamin D status in enrolled patients did not differ by gender, dosing group, diet, or diagnosis (Crohn disease or ulcerative colitis). Subjects reached steady-state serum 25OHD levels after three doses administered over a span of 4 to 8 months, our data demonstrated an increase in average 25OH vitamin D levels from 21.17 ng/mL to 28.19 ng/mL in the 50,000 IU and 23.00 ng/mL to 33.18 ng/mL in the 100,000 IU dose groups, respectively. The improvement in vitamin D status did not correlate with changes in quality of life or disease activity. The response to vitamin D therapy was independent of diet, sun exposure, race, gender, diagnosis, or season of enrollment. There were no adverse events, including changes in urine calcium to creatinine excretion or serum BUN and creatinine values. Several patients manifest a small decrease in serum phosphorus during the initial phase of the study. However, these changes were transient and no subjects exhibited clinical signs or symptoms of hypophosphatemia. CONCLUSION: High dose, interval vitamin D supplementation achieved steady-state 25OHD levels of 30 ng/mL or greater, with no signs of toxicity in patients enrolled in this pilot study. These data suggest that high-dose interval therapy may be a feasible treatment option that bypasses limitations related to difficulties with patient compliance. Further studies are necessary to determine optimal dosage regimens and to assess endpoints related to immune function and improvements to gastrointestinal health.

Doenças inflamatórias intestinais (DII) em humanos são reações crônicas de etiologia complexa. Trata-se de uma reação imunológica exacerbada e depende da microbiota. O sistema nervoso interage com a imunidade do intestino de um modo bidirecional. Relatos clínicos e poucos achados experimentais apontam para uma ligação entre transtornos depressivos e doenças inflamatórias intestinais, sugerindo interação neuroimunológica na patogenia deste processo. Ainda, o tratamento de Doenças inflamatórias intestinais (DIIDoença de Crohn e Colite Ulcerativa) com antidepressivos em modelos murino de colite têm sugerido bons resultados na redução da inflamação. O mecanismo da inflamação na DII e a participação do sistema nervoso ou da modulação de tal processo pelo emprego de antidepressivos ainda não está totalmente elucidado. Este estudo teve como objetivo estudar o efeito do antidepressivo amitriptilina em um modelo murino de colite. A colite foi induzida em camundongos C57BL/6 por Dextrano Sulfato de Sódio (DSS) e a amitriptilina (AMT) foi administrada por via oral, em regime profilático ou terapêutico. Avaliamos a dose de AMT no teste de suspensão da cauda (TSC), o acúmulo de neutrófilos pela atividade de mieloperoxidase (MPO), burst oxidativo, curva de sobrevida, histopatologia do intestino, atividade da doença por sintomas clínicos, a depleção de muco intestinal, citocinas inflamatórias no cólon e no soro, fenotipagem de linfócitos T CD4+, T CD8+, e monócitos CD14+,. Resultados: A dose de AMT (200 &mu;g/ml) e os regimes de tratamento utilizados aqui foram capazes de impedir ou diminuir a histopatologia da colite, os sinais clínicos (ganho de peso (%), comprimento e peso do cólon) e a mortalidade dos animais no modelo terapêutico do grupo inflamado e tratado com AMT. A atividade de MPO, níveis circulantes de IL- 1 &beta;, IL- 6 e TNF- &alpha; foram reduzidas nos dois protocolos experimentais (profilático e terapêutico). Conclusões: Este estudo incluiu um período de tratamento prolongado, visto que os antidepressivos são conhecidos por serem eficazes em seres humanos depois de várias semanas a meses de prescrição, e confirmou a eficiência da via de administração oral, uma vez que os antidepressivos são geralmente administrados por via oral a seres humanos. Este regime de tratamento melhorou o potencial anti-inflamatório de AMT na redução DSS-colite em camundongos, com base nos parâmetros estudados / Inflammatory bowel disease (IBD) in humans is a complex etiology of chronic reactions. It is an exacerbated immune reaction and depends on the microflora. The nervous system interacts with the intestinal immunity of a bidirectional fashion. Clinical reports and few experimental findings point to a link between depressive disorders and inflammatory bowel disease, suggesting neuroimmunological interaction in the pathogenesis of this process. Also, treatment of inflammatory bowel diseases (Crohn\'s disease DII- and Ulcerative Colitis) with antidepressants in murine models of colitis have pointed to positive results in reducing inflammation. The mechanism of inflammation in IBD and the involvement of the nervous system or modulation of this process by the use of antidepressants is not yet fully elucidated. This study aimed to study the effect of amitriptyline in a murine model of colitis. Colitis was induced in C57BL / 6 mice by Dextran Sodium Sulfate (DSS) and amitriptyline (AMT) were orally administered in a prophylactic or therapeutic regimen. We evaluated the AMT dose in the tail suspension test (TST), the accumulation of neutrophils by myeloperoxidase activity (MPO), oxidative burst, survival curve, bowel histopathology, disease activity by clinical symptoms, depletion of intestinal mucus, colon and inflammatory cytokines in the serum phenotype of CD4+ T lymphocytes, CD8+ and CD14+ monocytes. Results: The dose of AMT (200 &mu;g / ml) and treatment regimens used herein are able to prevent or decrease the pathology of colitis, clinical signs (weight gain (%), colon weight and length) and mortality animals in the therapeutic model inflamed group and treated with AMT. MPO activity, circulating levels of IL- 1 &beta;, IL- 6 and TNF- &alpha; were reduced in both experimental protocols (prophylactic and therapeutic). Conclusions: This study included a prolonged period of treatment, as antidepressants are known to be effective in humans after several weeks or months of limitation, and confirmed the effectiveness of oral administration route, since antidepressants are generally administered orally to humans. This treatment scheme has improved potential anti-inflammatory AMT in reducing DSS colitis in mice based on the study parameters

Inflammatory bowel disease (IBD) confers a high risk of development of colitis-associated colorectal cancer in patients with extensive colitis. Crypt fission is a mechanism of clonal expansion in the intestinal epithelium. Although fission is rare in the normal colon, many crypts in IBD patients are in the process of fission. Protumourigenic mutations can spread through the entire inflamed colon relatively quickly indicating that stem cell dynamics are altered in IBD. Some patients with IBD develop pseudopolyps as a result of mucosal ulceration and epithelial regeneration. The aim of this PhD was to investigate the effect of inflammation on niche succession, the crypt cycle and the expansion of clones in the IBD intestine. Pseudopolyps were examined as potential sites for clonal expansion by determining the frequency of mutated pseudopolyps and proliferative potential, and examining their microRNA (miRNA) profile relative to inactive, active and dysplastic mucosa, and adenoma and cancerous tissue. This thesis will show that crypt fission cycles in inflammatory bowel diseased colon are protracted and that each stage of crypt fission appears to be slow. Overall, clonally related adjacent IBD crypts seem to share a more recent common ancestor than non-related IBD crypts, supporting increased crypt fission rates in IBD. The proliferative drive induced by continuous inflammation and mucosal repair in ulcerative colitis (UC) appears to promote the expansion of CCO-deficient patches. Furthermore, niche succession appears to be faster in active IBD. Pseudopolyps are a source of regeneration within the epithelium and, as shown here, have a faster proliferative drive than background mucosa in IBD patients. Pseudopolyps are not genetically inert and are a potential source of protumourigenic mutations in UC. Hence, pseudopolyps are a potential reservoir within the inflamed epithelium where mutations are harboured and where there is no competition from neighbouring epithelium, as it has been denuded following previous inflammation. MiRNA expression in pseudopolyps differs from that of UC-dysplasia and mucosa. In particular, the MiR-29 family was downregulated in pseudopolyps, a miRNA family that has been implicated in intestinal fibrosis formation in stricturing Crohn’s disease. Pseudopolyps have been traditionally thought of as benign, genetically inert and incidental findings characteristic of chronic inflammation. My research runs counter to this view indicating an exciting paradigm shift in the way we consider pseudopolyps, which may eventually alter the endoscopic management of these lesions in the future.