Stefan Isaksson

T-Cell Activation in Patients With Irritable Bowel Syndrome

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 β7/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-1β, 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.

A Controlled Study of Colonic Immune Activity and β7+ Blood T Lymphocytes in Patients With Irritable Bowel Syndrome

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-gamma 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 beta7. 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 beta7+ was unaltered in IBS and UC patients, although the expression of mucosal addressin cell adhesion molecule-1+ endothelium, the ligand for integrin beta7, 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 beta7+ T lymphocytes in the peripheral blood. Our data further support the hypothesis of IBS being at least partially an inflammatory disorder.

The intra-individual variability of faecal calprotectin: a prospective study in patients with active ulcerative colitis.

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.

B‐cell activation in patients with irritable bowel syndrome (IBS)

Abstract  Patients with irritable bowel syndrome (IBS) may have a low grade immune activation. However, little is known about the properties of B cells of IBS patients. We therefore investigated activation level and antigen presenting phenotype of blood B cells of IBS patients. We also examined B‐cell responses to lipopolysaccharide (LPS) and probiotic bacteria. Blood samples were obtained from 74 IBS patients and 30 healthy subjects. Peripheral blood mononuclear cells were isolated and stimulated with LPS or an UV‐light inactivated bacterial cocktail consisting of the probiotic Gram‐positive strains; Lactobacillus paracasei ssp. paracasei 19, Lactobacillus acidophilus La5, Bifidobacterium lactis B612. The phenotype of CD19+ B cells was investigated by flow cytometry before and after 72 h cell culture. Furthermore, IBS symptom severity was assessed. B cells isolated from blood of IBS patients displayed an amplified activation level as demonstrated by increased cell surface expression of IgG, and also the costimulatory molecules CD80 and CD86. Expression of antigen presenting HLA‐DR and costimulatory molecule CD40 on B cells was, however comparable in IBS patients and controls. B cells of IBS patients displayed an impaired ability to increase expression of CD80, but not CD86, in response to both LPS as well as probiotic bacteria stimulations. To conclude, blood B cells of IBS patients have an increased activation level. Bacterial component induced expression of the costimulatory molecule CD80, regarded as important for tolerance induction, is impaired. These data suggest that B‐cell antigen presentation in IBS patients is associated with altered capacity of providing costimulation to T cells.

Altered levels of fecal chromogranins and secretogranins in IBS: relevance for pathophysiology and symptoms?

OBJECTIVES: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.METHODS: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.RESULTS:IBS patients demonstrated higher levels of fecal CgA (P=0.009), SgII (P<0.001), and SgIII (P<0.001), but lower levels of CgB (P<0.001) 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, P<0.001), SgII (r=−0.55, P<0.001), 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.CONCLUSIONS: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.

Fecal calprotectin one year after ileocaecal resection for Crohn's disease — A comparison with findings at ileocolonoscopy

BACKGROUND AND AIMS Ileocaecal resection for Crohns 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 Crohns 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 6months prior to the ileocolonoscopy. METHODS Thirty patients with Crohns 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 (>600μg/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 Crohns 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.

CD4 + CD25 + regulatory T cells in irritable bowel syndrome patients

Abstract  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+CD25low/− 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.

Fecal calprotectin levels predict the clinical course in patients with new onset of ulcerative colitis.

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 &mgr;g/g versus median, 102; IQR, 38–225 &mgr;g/g; P = 0.009) and 3 years of follow-up (median, 280; IQR, 102–622 &mgr;g/g versus median, 118; IQR, 39–219 &mgr;g/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 &mgr;g/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.

Serum IL-17A in Newly Diagnosed Treatment-Naive Patients with Ulcerative Colitis Reflects Clinical Disease Severity and Predicts the Course of Disease

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-&ggr; 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.

A pilot study of colonic B cell pattern in irritable bowel syndrome

Objective. 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. Material and methods. 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. Results. 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. Conclusions. 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.