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Background & Aims Intestinal macrophages adopt a hyporesponsive phenotype through education by local signals. Lack of proper macrophage maturation in patients with ulcerative colitis (UC) in remission may initiate gut inflammation. The aim, therefore, was to determine the effects of fecal luminal factors derived from healthy donors and UC patients in remission on macrophage phenotype and function. Methods Fecal supernatants (FS) were extracted from fecal samples of healthy subjects and UC patients in remission. Monocytes were matured into macrophages in the presence of granulocyte-macrophage colony-stimulating factor without/with FS, stimulated with lipopolysaccharide, and macrophage phenotype and function were assessed. Fecal metabolomic profiles were analyzed by gas-chromatography/mass-spectrometry. Results Fecal luminal factors derived from healthy donors were effective in down-regulating Toll-like receptor signaling, cytokine signaling, and antigen presentation in macrophages. Fecal luminal factors derived from UC patients in remission were less potent in inducing lipopolysaccharide hyporesponsiveness and modulating expression of genes involved in macrophage cytokine and Toll-like receptor signaling pathways. Although phagocytic and bactericidal abilities of macrophages were not affected by FS treatment, healthy FS-treated macrophages showed a greater ability to suppress cluster of differentiation 4+ T-cell activation and interferon γ secretion compared with UC remission FS-treated counterparts. Furthermore, metabolomic analysis showed differential fecal metabolite composition for healthy donors and UC patients in remission. Conclusions Our data indicate that UC patients in remission lack luminal signals able to condition macrophages toward a hyporesponsive and tolerogenic phenotype, which may contribute to their persistent vulnerability to relapse., Graphical abstract

Alteration of the host-microbiota cross talk at the intestinal barrier may participate in the pathophysiology of irritable bowel syndrome (IBS). Therefore, we aimed to determine effects of fecal luminal factors from IBS patients on the colonic epithelium using colonoids.Colon-derived organoid monolayers, colonoids, generated from a healthy subject, underwent stimulation with fecal supernatants from healthy subjects and IBS patients with predominant diarrhea, phosphate-buffered saline (PBS), or lipopolysaccharide (LPS). Cytokines in cell cultures and fecal LPS were measured by ELISA and mRNA gene expression of monolayers was analyzed using Qiagen RTColonoid monolayers stimulated with fecal supernatants from healthy subjects (n = 7), PBS (n = 4) or LPS (n = 3) presented distinct gene expression profiles, with some overlap (RFecal luminal factors from IBS patients induce a distinct colonic epithelial gene expression, potentially reflecting the disease pathophysiology. The culture of colonoids from healthy subjects with fecal supernatants from IBS patients may facilitate the exploration of IBS related intestinal micro-environmental and barrier interactions.

Background and Aims Alterations in the immunopathogenesis in ulcerative colitis [UC] during the disease course have been proposed. We therefore aimed to determine mucosal and systemic immune profiles in individual patients at the time of diagnosis [early disease] and after 10 years [late disease]. Methods Patients with UC provided serum and mucosal biopsies during a flare in early and in late disease. Serum samples were analysed using the Olink Proseek Inflammation panel. mRNA gene expression of biopsies was analysed using the Qiagen RT2 Profiler PCR Arrays Antibacterial response and T Helper Cell Differentiation. Results Orthogonal projections to latent structures discriminant analyses [OPLS-DA] demonstrated that the profile of 15 serum proteins discriminated in early and late disease [R2 = 0.84, Q2 = 0.65] in 15 UC patients. Eight of these proteins were differently expressed between the groups [Q Conclusions Mucosal and systemic immune profiles differ between early and late disease in patients with active UC, with a transition from a Th1- to a Th2-driven disease in the intestine. Improved understanding of the variation in immunopathogenesis during the disease course in UC is important to guide individualised treatment decision making.

Patients with ulcerative colitis (UC) have an altered gut microbiota composition, but the microbial relationship to disease activity needs to be further elucidated. Therefore, temporal dynamics of the fecal microbial community during remission and flare was determined. Fecal samples were collected at 2–6 time-points from UC patients during established disease (cohort EST) and at diagnosis (cohort NEW). Sampling range for cohort EST was 3–10 months and for cohort NEW 36 months. Relapses were monitored for an additional three years for cohort EST. Microbial composition was assessed by Genetic Analysis GA-map™ Dysbiosis test, targeting ≥ 300 bacteria. Eighteen patients in cohort EST (8 with maintained remission and 10 experiencing a flare), provided 71 fecal samples. In cohort NEW, 13 patients provided 49 fecal samples. The microbial composition showed no clustering related to disease activity in any cohort. Microbial dissimilarity was higher between than within patients for both cohorts, irrespective of presence of a flare. Microbial stability within patients was constant over time with no major shift in overall composition nor modification in the abundance of any specific species. Microbial composition was not affected by intensified medical treatment or linked to future disease course. Thus in UC, the gut microbiota is highly stable irrespective of disease stage, disease activity or treatment escalation. This suggests that prolonged dietary interventions or repeated fecal transplantations are needed to be able to induce permanent alterations of the gut microbiota.

BACKGROUND The clinical disease course of ulcerative colitis (UC) varies substantially between individuals and can currently not be reliably predicted. The gut microbiota and the host's immune defense are key players for gut homeostasis and may be linked to disease outcome. The aim of this study was to determine fecal microbiota composition and mucosal antibacterial response profile in untreated patients with newly diagnosed UC and the impact of these factors on disease course. METHODS Stool samples and intestinal biopsies were obtained from therapy-naive newly diagnosed patients with UC. Patients were defined to have mild or moderate/severe disease course assessed by disease activity during the 3 years follow-up. Fecal microbiota was analyzed by the GA-map Dysbiosis test (n = 18), and gene expression in intestinal biopsies was analyzed by RT Profiler polymerase chain reaction array (n = 13) and real-time polymerase chain reaction (n = 44). RESULTS At the time of diagnosis of UC, the fecal microbiota composition discriminated between patients with mild versus moderate/severe disease course. Also, the mucosal antibacterial gene expression response profile differed between patients with mild versus moderate/severe disease course with bactericidal/permeability-increasing protein (BPI) being most important for the discrimination. Mucosal bactericidal/permeability-increasing protein gene expression at diagnosis was higher in patients with mild versus moderate/severe disease course when confirmed in a larger patient cohort (P = 0.0004, n = 44) and was a good predictor for the number of flares during the 3 years follow-up (R = 0.395, P < 0.0001). CONCLUSIONS In patients with newly diagnosed UC, fecal microbiota composition and mucosal antibacterial response profile, especially bactericidal/permeability-increasing protein, are linked to disease course.

Evidence suggests that patients with irritable bowel syndrome (IBS) have an altered cytokine profile, although it is unclear whether cytokines are linked with symptom severity. We aimed to determine whether global serum and mucosal cytokine profiles differ between IBS patients and healthy subjects and whether cytokines are associated with IBS symptoms.Serum from 144 IBS patients and 42 healthy subjects was analyzed for cytokine levels of interleukin (IL)-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, interferon (IFN)-γ, and tumor necrosis factor (TNF) by MSD MULTI-ARRAY. In total, 109 IBS and 36 healthy sigmoid colon biopsies were analyzed for mRNA expression of IL-8, IL-10, TNF, and FOXP3 by quantitative reverse transcription PCR. Multivariate discrimination analysis evaluated global cytokine profiles. Rectal sensitivity, oroanal transit time, and psychological and gastrointestinal symptom severity were also assessed.Global cytokine profiles of IBS patients and healthy subjects overlapped, but cytokine levels varied more in IBS patients. Serum levels of IL-6 and IL-8 tended to be increased and levels of IFN-γ tended to be decreased in IBS patients. Mucosal mRNA expression of IL-10 and FOXP3 tended to be decreased in IBS patients. Within both the full study cohort and IBS patients alone, serum level of TNF was associated with looser stool pattern, while subjects with more widespread somatic symptoms had increased serum levels of IL-6. Although neither IBS bowel habit subgroups nor patients with possible post-infectious IBS were associated with distinct cytokine profiles, a small cluster of IBS patients with comparatively elevated immune markers was identified.Global cytokine profiles did not discriminate IBS patients from healthy subjects, but cytokine profiles were more varied among IBS patients than among healthy subjects, and a small subgroup of patients with enhanced immune activity was identified. Also, association of inflammatory cytokines with some clinical symptoms suggests that immune activation may be of importance in a subset of IBS patients.

Background and aims: The cellular mechanisms leading to infliximab therapy response in patients with ulcerative colitis (UC) are incompletely known. We therefore investigated early effects of infliximab therapy on monocytes and associated chemokines linked to clinical therapy response in UC patients. Methods: Blood and biopsies were obtained from anti-TNF therapy-naive UC patients ( n = 43) before (baseline) and during induction therapy with infliximab. Therapy response was evaluated at Week 14. Expression of monocyte activation markers and levels of chemokines in serum and biopsies were determined. Quantitative proteomic analysis was performed in cultured mucosal biopsies, and obtained data was validated in serum. Results: In therapy responders, but not in non-responders, infliximab reduced blood monocyte expression of CD14 and CD86, 2 weeks after therapy commenced, relative to baseline. Serum CCL2 levels were decreased only among therapy responders at Week 2 and Week 14, relative to baseline. These data corresponded with lower levels of CD14, CD86 and CCL2 in intestinal tissue in responders as compared with non-responders at Week 14. Proteomic analysis of cultured biopsies showed that infliximab induced a reduction in Tenascin C that predicted downregulation of CCL2. Therapy responders, but not non-responders, had decreased serum Tenascin C levels at Week 2 and Week 14, relative to baseline. Conclusions: Infliximab therapy response in UC patients is associated with reduced monocyte activation and serum levels of CCL2 2 weeks after therapy commencement. In therapy responders, infliximab influenced Tenascin C, which might be a regulator of CCL2 expression and important for induction of the clinical therapy response. * Abbreviations: : UC : ulcerative colitis IBD : inflammatory bowel disease TNF : tumor necrosis factor ECM : extracellular matrix TNC : Tenascin C PBMC : peripheral blood mononuclear cell MS : mass spectrometry IPA : ingenuity pathway analysis IPA KB : Ingenuity Pathways Analysis Knowledge Base MAP : molecule activity predictor

Background and Aims Ileocaecal resection for Crohn's disease is commonly performed. The severity of endoscopic lesions in the anastomotic area one year postoperatively is considered to reflect the subsequent clinical course. Fecal calprotectin (FC) has been shown to correlate with the findings at ileocolonoscopy in Crohn's disease. The objectives of this study were to assess whether the concentration of FC reflects the endoscopic findings one year after ileocaecal resection and to evaluate the variation of FC in individual patients during 6 months prior to the ileocolonoscopy. Methods Thirty patients with Crohn's disease and ileocaecal resection performed within one year were included. Stool samples were delivered monthly until an ileocolonoscopy was performed one year postoperatively. Results One year after surgery the median values of FC were not significantly different between the patients in endoscopic remission (n = 17) and the patients with an endoscopic recurrence (189 (75–364) vs 227 (120–1066)μg/g; p = 0.25). However, most patients with low values were in remission and all patients with high (> 600 μg/g) calprotectin values had recurrent disease. The variability of the FC concentration was most pronounced in patients with diarrhea. Conclusions We found no statistical difference in the concentrations of calprotectin between patients in endoscopic remission and patients with a recurrent disease one year after ileocaecal resection for Crohn's disease. However, among the minority of patients with low or high values, FC indicated remission and recurrence, respectively. There was significant variation of the fecal calprotectin concentrations over time, which affects the utility of calprotectin in clinical practice.

Background Little is known of the importance of chromogranins (Cg) and secretogranins (Sg) in ulcerative colitis (UC). We therefore investigated fecal levels of CgA, CgB, SgII and SgIII, and their association with inflammatory activity, disease duration and medical therapy in UC. Methods Analyses of CgA, CgB, SgII, SgIII and calprotectin in stool samples from 41 UC patients and 29 healthy controls were performed. Two stool samples, during relapse and remission, respectively, were obtained from each UC patient. Results The levels of fecal CgA and SgII were higher in UC patients with active disease as compared to healthy controls. CgB and SgII were positively correlated with disease duration, but none of the granins were positively correlated with calprotectin, Mayo score, CRP or serum concentrations of TNF in UC patients with active disease. Also UC patients in remission had higher levels of CgA, CgB, SgII, and SgIII as compared to healthy controls. However, levels of fecal CgA, CgB, SgII and SgIII were lower during active disease relative to remission. Moreover, fecal levels of CgA and SgII were higher in UC patients in remission treated with thiopurines than in thiopurine-naive patients in remission. Conclusion Fecal chromogranins and secretogranins are increased in UC but are not associated with disease activity, but seem to increase with duration of the disease. Thus, fecal granins might reflect structural changes associated with chronicity of disease, or medical therapy.

Background The clinical course of ulcerative colitis (UC) is unpredictable. The need for reliable biomarkers to reflect disease severity and predict disease course is therefore large. We investigated whether cytokines in mucosal tissue and serum reflect clinical disease severity at the onset of UC and predict the future disease course. Methods We prospectively monitored 102 patients from the onset of UC during 3 years, and they were followed up yearly for clinical and biochemical disease severity. Rectal biopsies were obtained from healthy controls and patients with UC. Serum and stool samples were obtained from patients with UC. Total mRNA from biopsies was analyzed with real-time PCR. Cytokine levels in serum were determined using Luminex or ELISA. Results Mucosal mRNA expression of IL-17A was 99.8 times higher while IFN-γ and IL-13 expression was increased 12.4 and 6.7 times, respectively, in patients relative to controls. Serum IL-17A correlated with clinical disease severity at the onset. Also, contrary to a number of other parameters, serum IL-17A at the onset predicted the clinical and biochemical course of the disease, as reflected by the Mayo score, number × severity of flares, and fecal calprotectin levels, respectively, during 3 years after the onset of the disease. None of these associations were found with mucosal cytokines at the onset. Conclusions Serum IL-17A levels of treatment-naive patients with UC reflect clinical disease severity at the onset of the disease and also predicted the disease course over the following 3 years. Thus, serum IL-17A may be valuable in the clinical management of patients with UC at the onset of the disease.

Many patients with inflammatory bowel disease (IBD) are undergoing therapy with infliximab, an antibody specific for TNF. However, the exact mechanisms of action of infliximab are not completely understood. The aim of this study was to determine the in vitro effects of infliximab on blood T cells derived from anti-TNF therapy-naïve ulcerative colitis (UC) patients with clinically active disease. Peripheral blood mononuclear cells were stimulated polyclonally or by antigen in the presence or absence of infliximab. The T cell phenotype was investigated by flow cytometry, cytokine secretion was determined by ELISA, and cell proliferation was determined by thymidine assay or CFSE dye. Presence of infliximab resulted in reduced expression of CD25 in CD4(+) and CD8(+) T cell populations and inhibited secretion of IFN-γ, IL-13, IL-17A, TNF as well as granzyme A. Infliximab also suppressed CD4(+) and CD8(+) T cell proliferation. These effects of infliximab were recorded both in T cells activated by polyclonal and antigen-specific stimulation. The effects of infliximab on T cell apoptosis and induction of FOXP3(+) CD4(+) T regulatory cells were ambiguous and depended on the originating cellular source and/or the stimulation mode and strength. In conclusion, infliximab is able to reduce T cell activation as measured by CD25, proliferation and cytokine secretion in vitro from UC patients with clinically active disease. These data suggest that suppression of T cell activity may be important for infliximab-mediated disease remission in patients with UC.

Background The clinical course of ulcerative colitis (UC) is unpredictable. During recent years, the ability of fecal biomarkers to predict relapse in inflammatory bowel disease has been evaluated. The objective of this study was to assess fecal calprotectin (FC) as a predictor of disease recurrence in patients with new onset of UC. Methods Sixty-nine patients were included. After the initial treatment, patients were followed up after 3 months and then yearly for 3 years. The prognostic role of FC 3 months after the initial therapy was evaluated. Results The FC levels 3 months after the diagnosis were higher in patients experiencing a relapsing disease course compared with those with a mild disease course during 1 year (median, 263; interquartile range [IQR], 100-634 μg/g versus median, 102; IQR, 38-225 μg/g; P = 0.009) and 3 years of follow-up (median, 280; IQR, 102-622 μg/g versus median, 118; IQR, 39-219 μg/g; P = 0.01). The area under the receiver operating characteristic curves using calprotectin to predict a relapsing disease course during 1 year and 3 years were 0.69 (95% confidence interval, 0.56-0.82) and 0.70 (95% confidence interval, 0.57-0.83), respectively. In the Kaplan-Meier survival analysis, a FC level >262 μg/g was associated with an increased risk of a relapsing disease course during the study period (P = 0.003). In logistic regression analysis, only FC and age were found to be independent predictors of having a relapsing disease course. Conclusions Levels of FC 3 months after the initial therapy in patients with new onset of UC predict the disease course over the following years, and they are of value in the clinical management of these patients.

Chromogranins (Cg) and secretogranins (Sg) are proteins ubiquitous in secretory cells of the enteric, endocrine, and immune systems, and may reflect activity of these systems. We therefore performed a hypothesis generating study to evaluate the association between fecal levels of CgA, CgB, SgII, and SgIII, with the clinical and pathophysiological phenotype of irritable bowel syndrome (IBS) patients.Analyses of CgA, CgB, SgII, SgIII, and calprotectin in fecal samples of 82 IBS patients and 29 healthy controls were performed. All IBS subjects completed validated questionnaires to assess gastrointestinal and psychological symptom severity, and underwent rectal barostat test and colonic transit time measurement.IBS patients demonstrated higher levels of fecal CgA (P=0.009), SgII (P0.001), and SgIII (P0.001), but lower levels of CgB (P0.001) compared with controls. SgII had good discriminative validity to positively identify IBS patients, with an area under the receiver operating characteristics (ROC) curve (AUROC) of 0.86 (95% confidence interval (CI): 0.78-0.94). SgIII and CgB both had fairly good discriminative validity to positively identify IBS patients, with an AUROC of 0.79 (95% CI: 0.71-0.87) and 0.78 (95% CI: 0.69-0.87), respectively. There were negative correlations between the colonic transit time and fecal levels of CgA (r=-0.53, P0.001), SgII (r=-0.55, P0.001), and SgIII (r=-0.28, P=0.03). Perceived abdominal pain was moderately associated with levels of CgA (r=0.32, P=0.004), SgII (r=0.31, P=0.006), and SgIII (r=0.24, P=0.04). Calprotectin levels were not associated with the levels of granins or with the clinical or pathophysiological phenotype of IBS patients.Fecal levels of Cg and Sg may be related to the underlying pathophysiology of IBS and of potential importance for symptoms of the patients. Granins also show promise to serve as future biomarkers of IBS. Further studies are needed to explore the potential role of granins in IBS patients.

The aetiology of the irritable bowel syndrome (IBS) is incompletely understood. A low-grade colonic inflammation is frequently seen, but it is unclear to what extent this phenomenon contributes to the pathophysiology of IBS. CD4(+)CD25(+) regulatory T cells (Treg) are implicated to play an important role in suppressing intestinal inflammation. We, therefore, examined whether the intestinal inflammatory process in IBS patients is the result of an altered function and/or frequency of CD25(+) Treg cells. Patients with IBS (n = 34), fulfilling the Rome II criteria, were compared with controls (n = 26). The suppressive activity of blood CD25(+) Treg cells was determined and the frequency of colonic and blood CD25(+) Treg cells was analysed by flow cytometry. The expression of the Treg marker, FOXP3 mRNA, in colonic biopsies was determined by reverse transcription-polymerase chain reaction. Blood CD25(+) Treg cells from IBS patients suppressed the proliferation of blood CD4(+)CD25(low/-) T cells. Similar frequencies of CD25(+) Treg cells were recorded in mucosa and blood of IBS patients and controls. FOXP3 mRNA was equally expressed in the colonic mucosa of patients with IBS and controls. In conclusion, the low-grade intestinal inflammation recorded in patients with IBS is not associated with an altered function or frequency of CD25(+) Treg cells.

Summary Background Anti-tumour necrosis factor (TNF) therapy is used for treatment of ulcerative colitis (UC). As approximately 30% of patients with UC do not benefit from the treatment, it is of clinical interest to identify biomarkers of response before therapy is initiated. Aim To identify prognostic biomarkers of anti-TNF therapy response in anti-TNF therapy-naive patients with UC. Methods Peripheral blood cells were obtained from 56 patients with UC before therapy started. Thirty-four patients were included in an exploratory cohort and 22 patients in a validation cohort. Blood cells were stimulated in vitro with influenza vaccine with and without anti-TNF. T-cell surface receptor expression and cytokine release were determined (in total 17 variables). Treatment response was evaluated using the Mayo score 12–14 weeks after the first infusion. Results In the exploratory cohort, blood cells from the patients showed stronger anti-TNF-dependent suppression of T-cell surface receptor expression and cytokine secretion among therapy responders than nonresponders. In particular, anti-TNF suppressed the expression of CD25 on T cells and secretion of interleukin 5, to a higher degree in responders than in nonresponders. These variables were used to a create model to predict therapy outcome, which was confirmed in the validation cohort. Correct classification of future therapy response was achieved in 91% of the cases in the validation cohort. Conclusion The effects of anti-TNF on cultured blood T cells, obtained before therapy started, predict treatment outcome in patients with UC.

Targeted therapy, using biomarkers to assess disease activity in ulcerative colitis (UC), has been proposed.The objective of this study was to evaluate whether pharmacological intervention guided by fecal calprotectin (FC) prolongs remission in patients with UC.A total of 91 adults with UC in remission were randomized to an intervention group or a control group. Analysis of FC was performed monthly, during 18 months. A FC value of 300 µg/g was set as the cut-off for intervention, which was a dose escalation of the oral 5-aminosalicylate (5-ASA) agent. The primary study end-point was the number of patients to have relapsed by month 18.There were relapses in 18 (35.3%) and 20 (50.0%) patients in the intervention and the control groups, respectively (p = 0.23); and 28 (54.9%) patients in the intervention group and 28 (70.0%) patients in the control group had a FC 300 µg/g, of which 8 (28.6%) and 16 (57.1%) relapsed, respectively (p 0.05).Active intervention significantly reduced relapse rates, although no significant difference was reached between the groups overall. Thus, FC-levels might be used to identify patients with UC at risk for a flare, and a dose escalation of their 5-ASA agent is a therapeutic option for these patients.

Background and aims: Leukocyte-derived proteins in faeces, especially calprotectin, are increasingly used to assess disease activity in ulcerative colitis. The objectives of the present study were to assess the importance of factors related to the stool sampling procedure. Methods: For 2 days, patients with active ulcerative colitis collected two stool samples at each bowel movement. The time of defecation, consistency and presence of blood were self-recorded in a diary. The variability in the concentrations of calprotectin during the day and between two consecutive days was assessed, as was the stability of calprotectin concentrations in samples stored at room temperature. Results: Altogether, 18 patients collected 287 stool samples. The intraclass correlation coefficient in pairs of samples from 132 bowel movements was 0.79 (95% CI 0.48–0.90). The median individual coefficient of variation in samples collected during the same day was 52% (4–178). There was a correlation between the level of calprotectin and the time between bowel movements (r = 0.5; p = 0.013). After 3 days at room temperature the calprotectin concentrations in stool samples were unchanged, but after 7 days a significant (p < 0.01) decrease was found (mean 28%; 95% CI 0.10–0.47). Conclusion: The present data reveal a great variability in the concentrations of calprotectin in stool samples collected during a single day. Since the levels of calprotectin increased with longer time between the bowel movements, it seems most appropriate to analyse stool from the first bowel movement in the morning. Moreover, storage of stool samples at room temperature for more than 3 days is not advisable. * Abbreviations : CAI : Clinical Activity Index SD : standard deviation ICC : intraclass correlation coefficient CV : coefficient of variation.

OBJECTIVES The understanding of the mechanisms for increased immune activation in subgroups of patients with irritable bowel syndrome (IBS) is incomplete. We hypothesized that monocytes are more activated in patients with IBS than in the healthy population. We therefore examined activation phenotype and cytokine secretion of blood monocytes. METHODS Blood samples from 74 patients with IBS and 30 controls were obtained. The activation phenotype of CD11cCD14 monocytes and cytokine secretion in serum and in peripheral blood mononuclear cells cultured with or without lipopolysaccharide was determined. Gastrointestinal and psychological symptom severity and quality of life were assessed using validated questionnaires. RESULTS Monocytes from patients demonstrated an increased expression of toll-like receptor (TLR) 2, whereas the expression on monocytes of TLR4, HLA-DR, CD40, CD80 and CD86 was comparable in patients and controls. The expression of activation markers on monocytes did not correlate with gastrointestinal or extracolonic symptom severity, but the expressions of TLR2, HLA-DR and CD86 were associated with less severe psychological symptoms and better social and physical well-being. Cytokine secretion in serum and peripheral blood mononuclear cell cultures was comparable in patients and controls. A subgroup of patients (15%) who had TLR2 and HLA-DR expression intensity above the level seen in controls reported less severe psychosocial symptoms. CONCLUSION Patients with IBS have increased expression of TLR2 on monocytes and the activation level on monocytes correlates with less severe psychological symptoms and better quality of life. Thus, our data implicate less importance of psychosocial factors and increased importance of immunological parameters for symptom generation in a subgroup of patients with IBS.

OBJECTIVES: Irritable bowel syndrome (IBS) has been found to be associated with low-grade immune activation in a subset of patients. We therefore investigated blood and colonic T-cell activity in IBS patients. METHODS: Blood samples were initially obtained from 74 IBS patients and 30 controls. Supplementary blood samples, to confirm data, were taken from another cohort (26 patients and 14 controls). In addition, colonic biopsies were taken from a third cohort (11 patients and 10 controls). Peripheral blood and colonic mononuclear cells were stimulated with anti-CD3/CD28 antibodies. Proliferation, cytokine secretion, and T-cell phenotype were investigated. IBS symptom severity was assessed. RESULTS: IBS patients displayed an activated phenotype with increased frequencies of blood T cells expressing CD69 and integrin beta7/HLA-DR. Anti-CD3/CD28-stimulated blood and colonic T cells from IBS patients proliferated less than T cells from controls. IBS patients had an increased polyclonally stimulated T-cell secretion of IL-1beta, which also weakly correlated with increased bowel habit dissatisfaction. Furthermore, despite normal frequencies of CD25high T cells in the blood of IBS patients, lower blood CD25high T-cell frequencies were modestly correlated with more bowel habit dissatisfaction and increased total IBS symptom severity. CONCLUSIONS: IBS patients have an increased frequency of activated T cells, demonstrated by the expression of activation markers and reduced proliferation in response to restimulation in vitro. The increased level of T-cell activation is consistent with the hypothesis of low-grade immune activation in IBS and may also be involved in symptom generation in IBS.

Patients with irritable bowel syndrome (IBS)may have a low grade immune activation. However,little is known about the properties of B cells of IBSpatients.Wethereforeinvestigatedactivationlevelandantigen presenting phenotype of blood B cells of IBSpatients. We also examined B-cell responses tolipopolysaccharide (LPS) and probiotic bacteria. Bloodsamples were obtained from 74 IBS patients and 30healthy subjects. Peripheral blood mononuclear cellswere isolated and stimulated with LPS or an UV-lightinactivated bacterial cocktail consisting of the probi-oticGram-positivestrains;Lactobacillusparacaseissp.paracasei 19, Lactobacillus acidophilus La5, Bifido-bacterium lactis B612. The phenotype of CD19 + B cellswas investigated by flow cytometry before and after72 h cell culture. Furthermore, IBS symptom severitywas assessed. B cells isolated from blood of IBSpatients displayed an amplified activation level asdemonstrated by increased cell surface expression ofIgG, and also the costimulatory molecules CD80 andCD86. Expression of antigen presenting HLA-DR andcostimulatory molecule CD40 on B cells was, howevercomparable in IBS patients and controls. B cells of IBSpatients displayed an impaired ability to increaseexpression of CD80, but not CD86, in response to bothLPS as well as probiotic bacteria stimulations. Toconclude, blood B cells of IBS patients have anincreased activation level. Bacterial componentinduced expression of the costimulatory moleculeCD80, regarded as important for tolerance induction,is impaired. These data suggest that B-cell antigenpresentation in IBS patients is associated with alteredcapacity of providing costimulation to T cells.Keywords immune activation, irritable bowelsyndrome, lipopolysaccharide, lymphocyte, probiotics.

Low-grade gastrointestinal inflammation has been reported in patients with irritable bowel syndrome (IBS). However, the colonic B-cell pattern has not been investigated in these patients. Therefore, the aim of this pilot study was to investigate the distribution and isotype of immunoglobulin-producing B cells in the colonic mucosa of IBS patients.Patients with IBS (n=12) fulfilling the Rome II criteria were compared with controls (n=11). Immunohistochemical staining of biopsies from the sigmoid and ascending colon was performed.The number of IgA(+) B cells in the ascending colon was lower in IBS patients than in controls (p=0.039). Furthermore, unlike controls, IBS patients had a reduction of IgA(+) B cells in the ascending colon relative to the sigmoid colon (p=0.04). Neither the IgG(+), nor the IgM(+) colonic B-cell numbers differed between IBS patients and controls. Very few colonic IgE(+) cells were detected and there was no difference between the two subject groups.The reduced number of colonic IgA(+) B cells in IBS patients suggests that the disorder may be associated with a modified gut immune defence. Whether this phenomenon is causally related to symptoms remains unknown and merits further investigation in a larger group of patients.

Background & Aims: The mechanisms behind irritable bowel syndrome (IBS) are incompletely understood. Recently several studies have suggested a low-grade colonic inflammation as initiator of the gut dysfunctions recorded in this patient group. The aim of this study was to characterize the phenotype and homing properties of colonic and peripheral blood lymphocytes in patients with IBS. Methods: Patients with IBS (n = 33), defined by the Rome II criteria, were compared with UC patients (n = 23) and control subjects (n = 15) without gastrointestinal symptoms. Colonic and peripheral blood lymphocytes were analyzed by flow cytometry. Secretion of IFN-γ from intestinal biopsies was determined by enzyme-linked immunosorbent assay, and immunohistochemical staining of colonic biopsies was performed. Results: IBS patients displayed an increased frequency of peripheral blood CD4+ and CD8+ T cells expressing the gut homing integrin β7. Accordingly, IBS and UC patients had an augmented frequency of lamina propria CD8+ T cells in the ascending colon as compared with control subjects. The frequency of intestinal T cells expressing integrin β7+ was unaltered in IBS and UC patients, although the expression of mucosal addressin cell adhesion molecule–1+ endothelium, the ligand for integrin β7, was increased in the ascending colon of IBS and UC patients as compared with control subjects. Conclusions: Patients with IBS exhibit an enhanced immune activity in the gut and an increased frequency of integrin β7+ T lymphocytes in the peripheral blood. Our data further support the hypothesis of IBS being at least partially an inflammatory disorder.

inflammatory bowel disease (IBD). Only limited data are available about the diagnostic accuracy of fecal matrix metalloprotease (MMP)-9 in IBD. The aims of our prospective study was to assess the diagnostic accuracy of fecal MMP-9 in patients with active Crohn’s disease (CD), ulcerative colitis (UC) and pouchitis assessed by clinical, endoscopic and histological scores and to compare the diagnostic accuracy of fecal MMP-9 and fecal calprotectin (CP) in IBD. Methods: Stool and blood samples were collected in 52 CD, 34 UC and 33 ileal pouch-anal anastomosis patients before control endoscopy. Biopsies were taken for histology. The activities of CD, UC and pouchitis were defined with the use of clinical, endoscopic and histological activity scores (CDAI, partial Mayo score, PDAI, SES-CD, Mayo endoscopic subscore, D’Haens and Riley score). Fecal CP and MMP-9 levels were quantified by use of enzyme-linked immunosorbent assay. Results: Active CD, UC and pouchitis was detected in 33.9%, 58.8% and 30.3% of the patients. Significant correlation was shown between the clinical, endoscopic and histological activities and fecal MMP-9 concentration in UC and pouchitis. Fecal CP concentration did not correlate with the clinical, endoscopic and histological activities of either UC or pouchitis. In CD, fecal CP correlated with the clinical, endoscopic and histological activities of the disease, and although fecal MMP9 concentration did not show significant correlation with SES-CD, stronger correlation was observed than in case of CP considering the clinical and histological activities of CD. Conclusions: This is the first study assessing the diagnostic accuracy of MMP-9 in different types of IBD. Our results showed that fecal MMP-9 has an exclusively high specificity in the detection of active UC and pouchitis and it also correlates stronger than fecal CP with the clinical and histological activities of CD. Further studies are needed to confirm these results.