Background: There is an unacceptable delay in diagnosis of axial Spondyloarthritis (axSpA) especially in its early stages among patients with inflammatory bowel disease (IBD). Objective: to develop a sensible and reliable questionnaire to identify undetected axSpA among IBD patients. Methods: Candidate items for the questionnaire were selected on 3 domains (IBD, inflammatory back symptoms, and extra-axial features). Sensibility of the Toronto axSpA Questionnaire (TASQ) was assessed leading to drafting 18 items. Test-retest reliability study was conducted among 77 patients with established IBD and axSpA and kappa agreement coefficients were calculated for items. Results: The TASQ was developed using multiple steps of sensibility assessment resulting in 16 items. Kappa coefficients ranged from 0.81 to 1.00 for all items indicating almost perfect agreement. Conclusion: TASQ is a newly developed, sensible and reliable questionnaire that should facilitate identification and referral of IBD patients to rheumatologists and should avoid delay in diagnosis of axSpA.

Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.

Inflammatory bowel disease (IBD; Crohn’s disease & ulcerative colitis) is a chronic illness in which medication and dietary adherence may determine disease natural history and severity of symptoms. We hypothesized that age, prospective memory (PM) and body satisfaction would predict medication and dietary adherence in adolescents with IBD and that gender and age would modify the relation between body satisfaction and adherence, with older girls being less adherent than younger children. Fifty-seven participants aged 10-21 (M = 16.5, SD = 2.3) with IBD and their caregivers were recruited. Informed consent, demographics and body satisfaction questionnaires were completed. PM was assessed using a naturalistic task. Adherence was measured by the 1-week completion of a medication and dietary log. A questionnaire was administered to evaluate coping strategies used for overcoming obstacles to dietary adherence. Two hierarchical regressions were conducted for medication and diet adherence respectively. As hypothesized, age had a significant effect (â = -.42, p < .01) on dietary adherence, accounting for approximately 17% of the variance (R2change = .17; Fchange (1,41) = 8.57, p = .006), with younger children being more adherent. Body satisfaction had a greater and more significant effect on dietary adherence than age (â = -.33, p < .01); i.e. participants more satisfied with their body reported better dietary adherence (R2change = .28; Fchange (2,35) = 6.97, p < .05). Findings remained consistent across multiple measures of body satisfaction and dietary adherence. None of the predictors had a significant effect on medication adherence. Health care providers who treat adolescents with IBD and parents should be made aware of factors affecting adherence in order to improve disease outcomes and patients’ quality of life.

Background: Increased numbers of intestinal E. coli are observed in inflammatory bowel disease, but the reasons for this proliferation and it exact role in intestinal inflammation are unknown. Aim of this PhD-project was to identify E. coli proteins involved in E. coli’s adaptation to the inflammatory conditions in the gut and to investigate whether these factors affect the host. Furthermore, the molecular basis for strain-specific differences between probiotic and harmful E. coli in their response to intestinal inflammation was investigated. Methods: Using mice monoassociated either with the adherent-invasive E. coli (AIEC) strain UNC or the probiotic E. coli Nissle, two different mouse models of intestinal inflammation were analysed: On the one hand, severe inflammation was induced by treating mice with 3.5% dextran sodium sulphate (DSS). On the other hand, a very mild intestinal inflammation was generated by associating interleukin 10-deficient (IL-10-/-) mice with E. coli. Differentially expressed proteins in the E. coli strains collected from caecal contents of these mice were identified by two-dimensional fluorescence difference gel electrophoresis. Results DSS-experiment: All DSS-treated mice revealed signs of a moderate caecal and a severe colonic inflammation. However, mice monoassociated with E. coli Nissle were less affected. In both E. coli strains, acute inflammation led to a downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower caecal concentrations of bacterial fermentation products than the control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterised protein YggE. NfuA was upregulated nearly 3-fold in both E. coli strains after DSS administration. Reactive oxygen species produced during intestinal inflammation damage Fe-S clusters and thereby lead to an inactivation of Fe-S proteins. In vitro data indicated that the repair of Fe-S proteins by NfuA is a central mechanism in E. coli to survive oxidative stress. Expression of YggE, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC under control and inflammatory conditions. In vitro growth experiments confirmed these results, indicating that E. coli Nissle is better equipped to cope with oxidative stress than E. coli UNC. Additionally, E. coli Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold higher than E. coli UNC. In turn, caecal indole concentrations resulting from cleavage of tryptophan by TnaA were higher in E. coli Nissle- associated control mice than in the respective mice associated with E. coli UNC. Because of its anti-inflammatory effect, indole is hypothesised to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle. Results IL-10-/--experiment: Only IL-10-/- mice monoassociated with E. coli UNC for 8 weeks exhibited signs of a very mild caecal inflammation. In agreement with this weak inflammation, the variations in the bacterial proteome were small. Similar to the DSS-experiment, proteins downregulated by inflammation belong mainly to the central energy metabolism. In contrast to the DSS-experiment, no upregulation of chaperone proteins and NfuA were observed, indicating that these are strategies to overcome adverse effects of strong intestinal inflammation. The inhibitor of vertebrate C-type lysozyme, Ivy, was 2- to 3-fold upregulated on mRNA and protein level in E. coli Nissle in comparison to E. coli UNC isolated from IL-10-/- mice. By overexpressing ivy, it was demonstrated in vitro that Ivy contributes to a higher lysozyme resistance observed for E. coli Nissle, supporting the role of Ivy as a potential fitness factor in this E. coli strain. Conclusions: The results of this PhD-study demonstrate that intestinal bacteria sense even minimal changes in the health status of the host. While some bacterial adaptations to the inflammatory conditions are equal in response to strong and mild intestinal inflammation, other reactions are unique to a specific disease state. In addition, probiotic and colitogenic E. coli differ in their response to the intestinal inflammation and thereby may influence the host in different ways. / Hintergrund: Chronisch entzündliche Darmerkrankungen zeichnen sich unter anderem durch eine starke Proliferation intestinaler E. coli aus. Unbekannt ist jedoch, ob diese Vermehrung eine Ursache oder eine Folge der Erkrankung darstellt. Ziel der vorliegenden Doktorarbeit war es daher, E. coli-Proteine zu identifizieren, welche der Anpassung an die entzündlichen Bedingungen im Darmtrakt dienen und unter Umständen einen Effekt auf den Gesundheitszustand des Wirtes haben. Weiterhin sollten die molekularen Ursachen für stammesspezifische Unterschiede zwischen probiotischen und gesundheitsschädlichen E. coli näher untersucht werden. Methoden: In den tierexperimentellen Analysen wurden keimfreie Mäuse entweder mit dem probiotischen E. coli Nissle oder dem adhärent-invasiven E. coli UNC monoassoziiert und in zwei verschiedenen Entzündungsmodellen näher untersucht. Einerseits wurde eine starke Darmentzündung durch die Gabe von 3,5% Natrium-Dextransulfat (DSS) ausgelöst. Andererseits wurde in Interleukin 10-defizienten (IL-10-/-) Mäusen eine sehr milde Form der Entzündung durch Besiedlung mit E. coli induziert. Die E. coli Bakterien wurden am Ende der Versuche aus den Caecuminhalten der Mäuse isoliert und die bakterielle Proteinexpression wurde mittels zwei-dimensionaler Gelelektrophorese analysiert. Ergebnisse des DSS-Versuchs: Alle Tiere des DSS-Versuchs entwickelten unabhängig vom E. coli Stamm, mit dem sie besiedelt waren, eine moderate Entzündung im Caecum und eine starke im Colon, wobei die Entzündungsreaktion durch die Monoassoziation mit E. coli Nissle leicht abgeschwächt wurde. In beiden E. coli Stämmen führte die Darmentzündung zu einer verringerten Expression von Enzymen des Kohlenhydratabbaus und der Energiegewinnung. In Folge dessen waren die intestinalen Konzentrationen bakterieller Fermentationsprodukte in den entzündeten Tieren geringer als in den gesunden Kontrolltieren. Weitere differentiell exprimierte Proteine umfassen das Fe-S- Cluster Reparaturprotein NfuA, die Tryptophanase TnaA und das uncharakterisierte Protein YggE. In beiden E. coli Stämmen, welche aus den DSS-Tieren isoliert wurden, war das NfuA Protein dreifach höher exprimiert. Eine Darmentzündung führt zu einer vermehrten Bildung reaktiver Sauerstoffspezies, welche die Fe-S-Cluster in Eisen-Schwefel-Proteinen zerstören und damit zu einer Inaktivierung dieser Proteine führen. In vitro Untersuchungen bestätigten, dass die Reparatur der Eisen-Schwefel-Proteine durch NfuA ein wichtiger Mechanismus ist um oxidativem Stress entgegenzuwirken. Das YggE Protein, welches laut Literaturangaben einen hemmenden Einfluss auf die Bildung reaktiver Sauerstoffspezies hat, war in E. coli Nissle 4- bis 8-fach erhöht (verglichen mit E. coli UNC unter Kontroll- und Entzündungsbedingungen). In vitro Versuche bestätigten diese Daten und zeigten, dass E. coli Nissle im Vergleich zu E. coli UNC eine erhöhte Resistenz gegenüber oxidativem Stress aufweist. Außerdem wurde im Vergleich E. coli Nissle vs. E. coli UNC (unter Entzündungs- und Kontrollbedingungen) ein 4- bis 7-fach erhöhter TnaA-Gehalt nachgewiesen. Indol, das Produkt der TnaA-katalysierten Tryptophanspaltung wurde in erhöhten Mengen im Intestinaltrakt E. coli Nissle-assoziierter Kontrolltiere detektiert. Seit längerem werden entzündungshemmende Eigenschaften für Indol postuliert, die aufgrund der Ergebnisse dieser Doktorarbeit nun auch mit den gesundheitsfördenden Eigenschaften von E. coli Nissle in Zusammenhang gebracht werden können. Ergebnisse des IL-10-/-- Versuchs: Nach einer 8-wöchigen Assoziationsdauer wurde nur in den mit E. coli UNC besiedelten IL-10-/- Tieren eine schwache Entzündungsreaktion nachgewiesen. Bedingt durch diese sehr schwach ausgeprägte Entzündungsantwort waren auch die Veränderungen im bakteriellen Proteom von E. coli UNC nur gering. Wie im DSS-Versuch waren Proteine des bakteriellen Energiestoffwechsels reprimiert, allerdings wurde keine Induktion von NfuA beobachtet. Daher scheint die Induktion von NfuA nur der Anpassung an eine starke Entzündung zu dienen. Weiterhin wurde nachgewiesen, dass E. coli Nissle aus IL-10-/- Tieren den Hemmer für das vertebrate C-Typ Lysozym (Ivy) sowohl auf mRNA- als auch auf Proteinebene stärker exprimiert als E. coli UNC. Überexpression von Ivy unter in vitro Bedingungen zeigte, dass es an der erhöhten Lysozymresistenz von E. coli Nissle beteiligt ist und somit eine Rolle als möglicher Fitnessfaktor von E. coli Nissle spielt. Schlussfolgerungen: In dieser Doktorarbeit wurde gezeigt, dass Darmentzündungen die Proteinexpression eines im Darm lebenden Bakteriums beeinflussen. Einige der aufgedeckten bakteriellen Anpassungsreaktionen werden sowohl bei einer starken als auch bei einer schwachen Entzündung ausgelöst; andere wiederum sind spezifisch für nur einen dieser Entzündungszustände. Weiterhin wurde deutlich, dass sich E. coli-Stämme hinsichtlich ihrer Reaktion auf eine Darmentzündung unterscheiden und damit möglicherweise den Wirt beeinflussen. 

This study investigated the association between flavonoid profiles of different cowpea (Vigna Unguiculata) varieties with anti-inflammatory properties as a possible benefit against inflammatory bowel disease. Cowpea, a drought tolerant annual herbaceous legume that originated in Africa, is known to possess high levels of polyphenolics, which have demonstrated anti-inflammatory, immunoregulatory and antioxidant properties. Black, red, white, brown and light brown cowpeas were investigated for phenolic content and composition using UV-Visible Spectroscopy and HPLC; antioxidant activation mechanism (AOX) by oxygen radical absorbance capacity (ORAC). Anti-inflammatory activity was measured via NF-κB activation in Raw 264.7 macrophages challenged with a lipo-polysaccharide. Phenols, tannins and AOX activity were generally similar within phenotypes; however among light brown varieties, 09FCV-CC-27M, had among the highest phenols, tannins and AOX, whereas IAR-48 had among the lowest. White cowpea (EARLY ACRE) variety showed the least amount of total phenol content (78.2 mg GAE/g) and condensed tannin content (4.1 mg CE/g); whereas the red varieties (IT82D-889, IT97K-1042-3) contained the highest amounts of tannins (242 and 132 mg CE/g), and phenols (431 and 454 mg GAE/g) respectively. Antioxidant activity correlated with phenol content data. Anthocyanins were only found in the black cowpea. The red varieties had the highest levels of flavonols, which were mostly quercetin derivatives; the white and light brown (IAR-48) varieties had quercetin-3-O-diglucoside as the dominantcompound. The light brown variety (09FCV-CC-27M) had the highest amount of flavan-3-ols while in the white variety no flavan-3-ols were detected. Unexpectedly, the cowpea extracts with lower phenolic and tannins content, the white and light brown (IAR-48) varieties, showed significant (p<0.05) anti-inflammatory properties in the LPS induced macrophages, inhibiting the activation of NF-κB at different concentrations (0.33, 1.67 and 3.33 μg extract/mL). Conversely, extracts with higher phenolic and tannin content did not induce anti-inflammatory response at similar concentrations, suggesting that tannins or other phenolics interfered with anti-inflammatory response at these concentrations. These results suggest that cowpea composition is an important determinant of anti-inflammatory response in inflammatory bowel disease.

Inflammatory bowel diseases (IBD) are associated with an elevated risk of colorectal cancer that increases with disease duration and severity. Tumor necrosis factor (TNF) is a major therapeutic target in IBD, but long-term anti-TNF therapy is associated with increased risks of infection and lymphoma, therefore we asked whether TNF signaling through its receptors TNFR1 and TNFR2 could impact colitis and colitis-associated cancer (CAC). In acute dextran sulphate sodium (DSS)-colitis, no major inflammatory differences were found between wildtype (WT), TNFR1- and TNFR2-deficient mice, with the exception of reduced macrophage infiltration into inflamed tissue in TNFR1-/- mice. Chronic colitis and tumor development was assessed in these mice using the carcinogen azoxymethane and 4 cycles of DSS. TNFR1-/- mice were protected against colorectal tumor development compared to WT and TNFR2-/- mice, while inflammation was similar between strains. Hematopoietic TNFR1 deficiency resulted in reduced inflammation and tumor incidence, while stromal/epithelial TNFR1 deficiency reduced indices of cancer without affecting inflammation. 8-OHDG was significantly lower in TNFR1-/- mice compared to other strains, suggesting that TNF could contribute to oxidative stress within the colon. Mice lacking leukocyte NADPH oxidase were protected against clinical illness and CAC despite similar histological inflammation, indicating that inflammation-associated oxidative stress can play a role in CAC. In conclusion, TNF signaling through TNFR1 contributes significantly to the development of colorectal cancer in a model of CAC in a manner that involves both stromal/epithelial and hematopoietic TNFR1. This is significant because anti-TNF therapies may be effective at reducing CAC in the absence of a clinical reduction of IBD symptoms.

This thesis explored the effects of cooked whole asparagus and its purified bioactive, rutin, on colitis symptoms and disease progression in mice using a chemically-induced model of colitis. This model mimics active colitis and recovery states of ulcerative colitis. C57BL/6 mice were fed a basal diet supplemented with 2% asparagus or 0.025% rutin for 3 weeks. Colitis was induced by 2% dextran sodium sulfate in drinking water for 7 days. Asparagus diet was determined to contain higher antioxidant capacities than rutin diet through antioxidant assays. During active colitis, consumption of asparagus alleviated some clinical symptoms (stool consistency, stool blood, and spleen hypertrophy) of colitis. In recovery, asparagus-fed mice were improving in terms of regenerating crypts, surface epithelial, and goblet cells, potentially due to its rutin content. Overall, these findings advocate that asparagus can be therapeutic in treating symptoms during active colitis and recovery phases of ulcerative colitis.

This study assesses generic and disease-specific Health-Related Quality of Life (HRQOL) in children and adolescents with Inflammatory Bowel Disease (IBD). More specifically, the purpose of the study is to address the relationship between disease- specific indicators, both on a symptom-by-symptom basis and as a whole, to overall HRQOL. Self- and proxy-report versions of the Pediatric Quality of Life Inventory™ (PedsQL™) Generic Core Scales and the newly developed Pediatric Quality of Life Inventory™ Gastrointestinal Symptoms Module were administered to 187 parent-child dyads at ten study sites across the United States. Disease-specific indicators included: stomach pain, stomach upset, trouble swallowing, heartburn and reflux, gas and bloating, constipation, and diarrhea. It was hypothesized that caregiver- and child-reported disease-specific HRQOL would be positively correlated with generic HRQOL, and that physical disease-specific indicators would contribute the greatest variance in total generic HRQOL scores, for both self and proxy report. Results confirmed the hypothesis that disease-specific HRQOL would be positively correlated with generic HRQOL for children and caregivers. Multivariate regression results revealed that the Stomach Pain and Hurt, Worry, Medicines, and Communication scales contributed the most variance to overall HRQOL scores for children. The same analysis performed for parent ratings yielded one statistically significant scale: Worry. In essence, intervention efforts aimed at reducing the influence of worry and anxiety may prove more effective in improving HRQOL outcomes than interventions targeting reduction of physical symptoms.

Inflammatory bowel disease (IBD) alters neuronal regulation of the gastrointestinal (GI) tract. The superior mesenteric ganglia (SMG) contain sympathetic neurons that modulate GI functions such, as motility and blood flow. IBD reduces the release of noradrenaline, a sympathetic neurotransmitter. We hypothesized that the reduction in NA release is due to inhibition of voltage-gated calcium current (ICa), as calcium influx is a regulator of neurotransmitter release. We also hypothesized that tumor necrosis factor α (TNFα), a proinflammatory cytokine elevated during IBD, can also inhibit the ICa of SMG neurons. Therefore, we compared ICa amplitude in neurons from normal mice and mice with dextran sulphate sodium (DSS; 5% w/v)-induced colitis. Neurons dissociated from the SMG were cultured overnight and changes to ICa were investigated using electrophysiological, Ca2+ imaging, PCR and neurotransmitter release techniques. Colitis significantly reduced ICa of SMG neurons by selectively inhibiting N-type Ca2+ channels. This was accompanied by a reduction in mRNA encoding the N-type channel alpha subunit (CaV 2.2) and a rightward shift in the voltage dependence of activation of ICa. Colitis reduced the NA release from the colon and jejunum. Depolarization-induced release of tritiated-NA was inhibited by ω-Conotoxin GVIA (300 nM). These results suggest that the changes in VGCC observed at the cell bodies of SMG neurons were also occurring at the nerve terminals during colitis. Similar experimental techniques were performed using SMG neurons incubated overnight in TNFα (1nM). TNFα decreased ICa and depolarization-induced Ca2+ influx in SMG neurons. Similar to DSS-induced colitis, the reduction in ICa was limited to N-type Ca2+ channels. Preincubation of neurons with SC 514 (20μM) and Bay 11 7082 (1µM), inhibitors of nuclear factor kappa B signaling, prevented the reduction in ICa. Preincubation with the p38 MAPK inhibitor, PD 169316 (30µM), recovered a smaller portion of the reduction in Ca2+ influx. These data suggest that DSS colitis and TNFα inhibit N-type VGCC ICa in sympathetic neurons and identify a novel role for NF-κB and p38 MAPK in the regulation of neurotransmitter release. These findings also suggest that DSS colitis inhibits NA release by altering sympathetic N-type VGCC in the colon and jejunum. / Thesis (Master, Physiology) -- Queen's University, 2008-09-02 12:06:20.438

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