In a population prevalence questionnaire study we demonstrated that constipation and fecal incontinence are common problems in the general Swedish population with a similar magnitude as in other Western countries. 95.6% of the population had between three bowel movements per day and three per week. Constipation was mostly defined by “hard stools” and “the need of using laxatives”. Irritable Bowel Syndrome (IBS) is characterized by abdominal pain/discomfort and abnormal bowel habits. The diagnostic criteria of IBS are based on clinical symptoms. Division of IBS patients into symptom subgroups appears important as their bowel symptoms are characterized by heterogeneity. International criteria to subgroup IBS (Rome II) are based on expert consensus and not on evidence. We investigated the variation of stool consistency and defecatory symptoms in 135 IBS patients by symptom diary cards. Most patients had alternating stool consistency. When subgroups were based on stool consistency, all kinds of defecatory symptoms (straining, urgency, and feeling of incomplete evacuations) were frequently present in all subgroups. Stool frequency was in the normal range in the majority of patients. We propose that IBS subgroups should be based on stool consistency. We suggest that Rome II supportive criteria must be reconsidered as the determination of presence or absence of specific symptoms does not work as an instrument for categorization of IBS patients into diarrhoea- and constipation-predominant. We also propose that abnormal stool frequency should be excluded to define subgroups of IBS. Alternating stool consistency and presence of different defecatory symptoms, regardless of stool consistency should be included as criteria for IBS. Stress is known to play an important role in the onset and modulation of IBS symptoms. From experimental studies there is evidence for a stress-dependent alteration of visceral sensitivity. The biological mechanisms responsible for the causal link between stress and IBS symptoms are not completely understood, but the hypothalamic-pituitary-adrenocortical axis and the autonomous nervous system seem to play a prominent role in the pathophysiology of IBS. We investigated visceral sensitivity and the effect of repeated maximal tolerable rectal distensions on salivary cortisol levels and skin conductance in patients with IBS, chronic constipation and healthy volunteers. We found that the expectancy of the experimental situation per se (provocation of bowel symptoms by rectal distensions) compared to non-experimental days at home measured as salivary cortisol had a high impact on the level of arousal in IBS. IBS patients had higher skin conductance values than controls in the beginning of distension series and lower rectal thresholds for first sensation, urge and discomfort than healthy controls and constipation patients. IBS patients demonstrated habituation to repeated subjective maximal tolerable rectal distensions according to sympathetic activity although patients continued to rate their discomfort as maximal. Constipation patients had lower sympathetic activity than IBS patients before and during repeated rectal distensions. None of the groups demonstrated a significant increase in cortisol after repetitive rectal distensions. We conclude that Rome II supportive criteria for IBS should be reconsidered according to our findings. IBS patients are more sensitive to pre-experimental stress than healthy controls and patients with constipation. This should be considered in the design of experimental IBS studies. IBS patients habituated to subjective maximal tolerable, repetitive rectal distensions with decreasing sympathetic activity. Since responses to repeated stimuli of close-to-pain intensities are resistant to habituation this finding could be caused by psychological influences on perception, that is, perceptual response bias.

Inflammatory bowel disease (IBD) comprises diseases that are characterized by chronic or relapsing inflammation of the gastrointestinal tract. Primary sclerosing cholangitis (PSC) is an extraintestinal manifestation in IBD. Immunoreactivity against an autoantigen that is expressed both in the gastrointestinal tract and the biliary tract could be the link between these diseases. A possible source of such an antigen is goblet cells. Immunostainings of normal human tissues using IBD patient sera showed goblet cell immunoreactivity against goblet cells in all parts of the gastrointestinal tract. The most frequent immunostaining was found against goblet cells in the appendix against which 84% (42/50) of IBD patients compared to 8% (4/50) of healthy blood donors showed immunoreactivity. To identify the corresponding antigen we used three different approaches, investigation of immunoreactivity to different candidate proteins compared to IBD sera, immunoscreening of an appendiceal cDNA library, and immunoprecipitation of protein lysates from mucin producing cells followed by SDS-PAGE and 2D gel electrophoresis. These approaches led to the identification of several candidate autoantigens of which complement C3 is the most promising. A novel staining pattern with strong immunoreactivity to granules and the apical membrane of biliary epithelial cells was identified with 35% (12/34) of PSC sera compared to none of healthy controls (n=28). Screening of a cDNA library from normal human choledochus identified PDZ domain containing 1 (Pdzk1) and Glutathion S transferase theta 1 (GSTT1) as potential candidates. Pdzk1 is an interesting candidate which is expressed in the intestinal tract and bile ducts. GSTT1 antibodies were not specific for PSC and are thought to develop as an alloimmune response in patients with the GSTT1-null genotype. In conclusion, we have identified specific immunoreactivity to goblet cells and biliary epithelial cells using sera from patients with IBD and PSC respectively. We have also identified several potential autoantigens.

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.

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. 

Hintergrund: Patienten mit chronischen entzündlichen Erkrankungen sind aufgrund ihrer Grundkrankheit, aber auch durch die häufig notwendige immunsuppressive Therapie gefährdet, an einer impfpräventablen Infektionskrankheit zu erkranken. In einer Stichprobe sollte der Impfstand bei Patienten mit chronisch-entzündlichen Darmerkrankungen (CED) erhoben werden. Besondere Beachtung galt Vorbehalten der Patienten gegen die empfohlenen Schutzimpfungen. Methoden: Wir baten 203 Patienten mit CED (davon 57% Mb. Crohn, 63% weiblich; medianes Alter 36 Jahre), die im letzten Jahr keine Impfberatung erhalten hatten, einen Fragebogen mit 38 Fragen zu beantworten. Zudem wurden alle Impfnachweise erfasst und mit den aktuellen Empfehlungen der Ständigen Impfkommission abgeglichen. Die Befragung erfolgte vom 1.4. bis 30.9.2009. Ergebnisse: Nur 83% der Patienten hatten einen Impfausweis. Es fanden sich erhebliche Impfdefizite; so wurden in den letzten zehn Jahren nur 67% der Patienten gegen Tetanus und 21% gegen Pertussis geimpft, 28% nahmen die Impfung gegen die saisonale Grippe 2008 wahr und nur 9% wurden jemals gegen Pneumokokken geimpft. Im Subgruppenvergleich von Patienten mit TNF-Blockern (n=39) mit denjenigen Patienten, die noch nie eine immunsuppressive Dauertherapie erhielten (n=67), zeigten sich keine Unterschiede. 80% der Patienten wären bereit, alle offiziell empfohlenen Schutzimpfungen durchführen zu lassen. 22% aller Patienten gaben an, Schutzimpfungen zu vermeiden, weil sie Nebenwirkungen befürchteten, 15% weil das Immunsystem „nicht intakt“ ist und 9% befürchten eine Verschlimmerung der CED durch eine Impfung. Schlussfolgerungen: Der Impfstand in der untersuchten Stichprobe war unzureichend. Es fand sich insbesondere eine deutliche Diskrepanz zwischen der hohen Bereitschaft der Patienten, Schutzimpfungen durchführen zu lassen, und dem tatsächlichen Impfstand. Unsere Daten legen die Notwendigkeit einer erhöhten ärztlicher Wachsamkeit für Impflücken bei immunsuppressiv behandelten Patienten nahe. / Background: Patients with chronic inflammatory diseases are at increased risk for vaccine preventable infectious diseases. This is caused by the inflammatory state itself as well as often necessary immunosuppressive therapy. In a random sample, we investigated whether patients with inflammatory bowel disease (IBD) are sufficiently vaccinated. Special attention was spent to arguments for vaccine refusal. Methods: Between 1.4.2009 and 30.9.2009, we asked 203 consecutive IBD patients (thereof 57% Crohn’s disease, 63% female; median age 36 years), who got no vaccination advise within the last year to answer a questionnaire with 38 questions. As well, the vaccination cards were adjusted with the official recommendations. Results: Only 83% of patients had a vaccination card. We recognized substantial vaccination deficiencies. Within the past 10 years, only 67% of patients had tetanus and 21% had pertussis vaccination. Only 28% had an influenza vaccination in 2008 and only 9% were ever immunized against pneumococcus. A subgroup analysis of patients with TNF-blockers (n=39) with patients, who never had immunosuppressive therapy (n=67) revealed no difference. 80% of all patients are willing to adhere to all officially recommended vaccinations. 22% and 15% of patients stated that they avoid vaccinations as they afraid side effects or as they assess their immune system as not intact. Nine per cent feared a worsening of IBD after vaccination. Conclusions: In this random sample, the adherence to vaccination recommendations was low. We observed a marked difference between the willingness of IBD patients for immunizations and the realized vaccinations. Our data suggest that an increased medical awareness for vaccination deficiencies in immunosuppressed patients is mandatory.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.