Abstract
Objective.
The intestinal microbiota plays a substantial role in the pathogenesis of inflammatory bowel disease (IBD). Faecalibacterium prausnitzii (FP) is underrepresented in IBD patients ...and have been suggested to have anti-inflammatory effects in mice. Increased intestinal permeability is common in IBD but the relationship between FP and intestinal barrier function has not been investigated. Our aim was to study treatment with FP supernatant on intestinal barrier function in a dextran sodium sulfate (DSS) colitis mice model.
Material and methods.
C57BL/6 mice received 3% DSS in tap water ad libitum during five days to induce colitis. From day 3 the mice received a daily gavage with FP supernatant or broth during seven days. Ileum and colon were mounted in Ussing chambers for permeability studies with 51Cr-EDTA and Escherichia coli K-12. Colon was saved for Western blot analyses of tight junction proteins.
Results.
DSS-treated mice showed significant weight loss and colon shortening. Gavage with FP supernatant resulted in a quicker recovery after DSS treatment and less extensive colonic shortening. Ileal mucosa of DSS mice showed a significant increase in 51Cr-EDTA-passage compared to controls. 51Cr-EDTA passage was significantly decreased in mice receiving FP supernatant. No significant differences were observed in passage of E. coli K12. Western blots showed a trend to increased claudin-1 and claudin-2 expressions in DSS mice.
Conclusions.
Supernatant of FP enhances the intestinal barrier function by affecting paracellular permeability, and may thereby attenuate the severity of DSS-induced colitis in mice. These findings suggest a potential role of FP in the treatment of IBD.
Altered intestinal barrier is associated with immune activation and clinical symptoms in diarrhoea-predominant IBS (IBS-D). Increased mucosal antigen load may induce specific responses; however, ...local antibody production and its contribution to IBS aetiopathogenesis remain undefined. This study evaluated the role of humoral activity in IBS-D.
A single mucosal jejunal biopsy, luminal content and blood were obtained from healthy volunteers (H; n=30) and IBS-D (n=49; Rome III criteria) participants. Intraepithelial lymphocytes, mast cells, B lymphocytes and plasma cells were studied by imaging techniques. Differential gene expression and pathway analysis were assessed by microarray and PCR techniques. Blood and luminal immunoglobulins (Igs) were quantified. Gastrointestinal symptoms, respiratory atopy and stress and depression were also recorded.
Patients with IBS-D showed a higher number and activation of mucosal B lymphocytes and plasma cells (p<0.05). Mast cell density was increased in patients with IBS-D (non-atopic) and in close proximity to plasma cells (p<0.05). Microarray profiling identified differential humoral activity in IBS-D, involving proliferation and activation of B lymphocytes and Igs production (p<0.001). Mucosal humoral activity was higher in IBS-D, with upregulation of germline transcripts and Ig genes (1.3-fold-1.7-fold increase; p<0.05), and increased IgG(+) cells and luminal IgG compared with H (p<0.05), with no differences in blood. Biological markers of humoral activity correlated positively with bowel movements, stool form and depression.
Enhanced small bowel humoral immunity is a distinctive feature of IBS-D. Mucosal Ig production contributes to local inflammation and clinical manifestations in IBS-D.
Background
Functional dyspepsia (FD) is a complex disorder, in which multiple mechanisms underlie symptom generation, including impaired duodenal barrier function. Moreover, an altered duodenal bile ...salt pool was recently discovered in patients with FD. We aimed to evaluate the relationship between bile salts, bacterial translocation, and duodenal mucosal permeability in FD.
Methods
Duodenal biopsies from patients with FD and healthy volunteers (HV) were mounted in Ussing chambers to measure mucosal resistance and bacterial passage in the absence and presence of fluorescein‐conjugated Escherichia coli and glyco‐ursodeoxycholic acid (GUDCA) exposure. In parallel, duodenal fluid aspirates were collected from patients and bile salts were analyzed.
Key results
The transepithelial electrical resistance of duodenal biopsies from patients was lower compared with HV (21.4 ± 1.3 Ω.cm2 vs. 24.4 ± 1.2 Ω.cm2; P = .02; N = 21). The ratio of glyco‐cholic and glyco‐chenodeoxycholic acid (GCDCA) to tauro‐ and GUDCA correlated positively with transepithelial electrical resistance in patients. Glyco‐ursodeoxycholic acid slightly altered the mucosal resistance, resulting in similar values between patient and healthy biopsies (22.1 ± 1.0 Ω.cm2 vs. 23.0 ± 1.0 Ω.cm2; P = .5). Bacterial passage after 120 minutes was lower for patient than for healthy biopsies (0.0 0.0‐681.8 vs. 1684.0 0.0‐4773.0 E coli units; P = .02). Glyco‐ursodeoxycholic acid increased bacterial passage in patient biopsies (102.1 0.0‐733.0 vs. 638.9 280.6‐2124.0 E coli units; P = .009). No correlation was found between mucosal resistance and bacterial passage.
Conclusions & inferences
Patients with FD displayed decreased duodenal mucosal resistance associated with bile salts, however, not associated with bacterial passage in vitro. In addition, the hydrophilic bile salt glyco‐ursodeoxycholic acid abolished differences in mucosal resistance and bacterial passage between patient and control group.
The duodenal mucosal transepithelial electrical resistance (TEER) was significantly lower in biopsies from functional dyspepsia patients compared to healthy controls. After exposure to glyco‐ursodeoxycholic acid, the TEER values between patient and HV biopsies were similar.
During acute pancreatitis (AP), oxidative stress contributes to intestinal barrier failure. We studied actions of multispecies probiotics on barrier dysfunction and oxidative stress in experimental ...AP.
Fifty-three male Spraque-Dawley rats were randomly allocated into five groups: 1) controls, non-operated, 2) sham-operated, 3) AP, 4) AP and probiotics and 5) AP and placebo. AP was induced by intraductal glycodeoxycholate infusion and intravenous cerulein (6 h). Daily probiotics or placebo were administered intragastrically, starting five days prior to AP. After cerulein infusion, ileal mucosa was collected for measurements of E. coli K12 and (51)Cr-EDTA passage in Ussing chambers. Tight junction proteins were investigated by confocal immunofluorescence imaging. Ileal mucosal apoptosis, lipid peroxidation, and glutathione levels were determined and glutamate-cysteine-ligase activity and expression were quantified. AP-induced barrier dysfunction was characterized by epithelial cell apoptosis and alterations of tight junction proteins (i.e. disruption of occludin and claudin-1 and up-regulation of claudin-2) and correlated with lipid peroxidation (r>0.8). Probiotic pre-treatment diminished the AP-induced increase in E. coli passage (probiotics 57.4+/-33.5 vs. placebo 223.7+/-93.7 a.u.; P<0.001), (51)Cr-EDTA flux (16.7+/-10.1 vs. 32.1+/-10.0 cm/s10(-6); P<0.005), apoptosis, lipid peroxidation (0.42+/-0.13 vs. 1.62+/-0.53 pmol MDA/mg protein; P<0.001), and prevented tight junction protein disruption. AP-induced decline in glutathione was not only prevented (14.33+/-1.47 vs. 8.82+/-1.30 nmol/mg protein, P<0.001), but probiotics even increased mucosal glutathione compared with sham rats (14.33+/-1.47 vs. 10.70+/-1.74 nmol/mg protein, P<0.001). Glutamate-cysteine-ligase activity, which is rate-limiting in glutathione biosynthesis, was enhanced in probiotic pre-treated animals (probiotics 2.88+/-1.21 vs. placebo 1.94+/-0.55 nmol/min/mg protein; P<0.05) coinciding with an increase in mRNA expression of glutamate-cysteine-ligase catalytic (GCLc) and modifier (GCLm) subunits.
Probiotic pre-treatment diminished AP-induced intestinal barrier dysfunction and prevented oxidative stress via mechanisms mainly involving mucosal glutathione biosynthesis.
Background & Aims Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and ...corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients. Methods Transmucosal fluxes of protein antigens (horseradish peroxidase) and paracellular markers (51 Cr-EDTA, fluorescein isothiocyanate–dextran 4000) were studied in noninflamed, colonic mucosal biopsy samples collected from 26 patients with UC and 53 healthy volunteers (controls); samples were mounted in Ussing chambers. We also performed fluorescence and electron microscopy of human tissue samples, assessed isolated eosinophils, and performed mechanistic studies using in vitro cocultured eosinophils (15HL-60), mast cells (HMC-1), and a colonic epithelial cell line (T84). Results Colon tissues from patients with UC had significant increases in permeability to protein antigens compared with controls. Permeability was blocked by atropine (a muscarinic receptor antagonist), α-helical CRF(9–41) (a CRF receptor antagonist), and lodoxamide (a mast-cell stabilizer). Eosinophils were increased in number in UC tissues (compared with controls), expressed the most M2 and M3 muscarinic receptors of any mucosal cell type, and had immunoreactivity to CRF. In coculture studies, carbachol activation of eosinophils caused production of CRF and activation of mast cells, which increased permeability of T84 epithelial cells to macromolecules. Conclusions We identified a neuroimmune intercellular circuit (from cholinergic nerves, via eosinophils to mast cells) that mediates colonic mucosal barrier dysfunction in patients with UC. This circuit might exacerbate mucosal inflammation.
: Clinical observations suggest that the sites of initial inflammation in ileal Crohn's disease (CD) are the lymphoid follicles, where the aphtoid lesions originate from small erosions of the ...follicle‐associated epithelium (FAE). Lymphoid follicles and Peyer's patches (PPs) consist of a number of B‐cell follicles with intervening T cell areas. The T cell follicular area is also populated by dendritic cells (DCs) and macrophages. A single layer of epithelial cells covering each follicle forms a dome between the surrounding villi. This FAE differs from normal villus epithelium in several ways that make the epithelial cells of the FAE more exposed to the luminal contents, more accessible to antigens, and in closer contact with the immune system. The most prominent feature is the presence of specialized M cells, which are optimized for antigen adherence and transport. M cells play an important role in the surveillance of the intestinal lumen, but also provide a route of entry for various pathogens. In this article we review the current knowledge on the epithelial phenotype of the human FAE, and changes of the FAE and M cells in intestinal inflammation, leading to a hypothesis of the role of the FAE and M cells in the pathogenesis of CD.
Functional dyspepsia (FD) is an extremely common functional gastrointestinal disorder, the pathophysiology of which is poorly understood. We hypothesised that impaired intestinal barrier function is ...involved in the onset and persistence of this disorder by inducing low-grade inflammation. Therefore, our aim was to evaluate duodenal mucosal integrity and low-grade inflammation in patients with FD.
Duodenal biopsy specimens were obtained from 15 patients with FD fulfilling the Rome III criteria and 15 age- and gender-matched healthy volunteers. Transepithelial electrical resistance (TEER) and paracellular permeability were measured in Ussing chambers. Expression of cell-to-cell adhesion proteins was evaluated by real-time PCR, western blot and/or immunofluorescence. Numbers of mast cells, eosinophils and intraepithelial lymphocytes were assessed by immunohistochemistry.
Patients with FD displayed lower TEER and increased paracellular passage compared with healthy controls, which is indicative of impaired mucosal integrity. In addition, abnormal expression of cell-to-cell adhesion proteins at the level of tight junctions, adherens junctions and desmosomes was shown. Furthermore, patients were characterised by the presence of low-grade inflammation, as demonstrated by increased infiltration of mucosal mast cells and eosinophils. A significant association between the expression level of several cell-to-cell adhesion proteins, the extent of increased permeability and the severity of low-grade inflammation was found.
These findings challenge the classical paradigm that patients with FD show no structural changes in the gastrointestinal tract. We suggest that impaired intestinal barrier function is a pathophysiological mechanism in FD. Thus, restoration of intestinal barrier integrity may be a potential therapeutic target for treating patients with FD.
Crohn's disease (CD) is characterized by overproduction of proinflammatory cytokines like interleukin (IL)-1beta. Production of mature IL-1beta is dependent on a caspase-1-activating protein complex ...called the NALP3 inflammasome, composed of NALP3, ASC, and CARD8. NALP3 shares structural similarities with Nod2, and both of these proteins are required for bacteria-induced IL-1beta secretion. The combination of the polymorphisms CARD8 (C10X)and NALP3 (Q705K) was recently shown to be associated with rheumatoid arthritis.Our aim was to investigate whether these combined polymorphisms play a role in the susceptibility to CD.
The study included 498 CD patients in two cohorts from different regions and 742 control individuals from a Swedish population. DNA was isolated from whole blood. Polymorphisms of (Q705K) NALP3 and (C10X) CARD8, as well as the Nod2 variants, R702W and G908R, were genotyped using the Taqman single nucleotide polymorphism assay. The Nod2 frameshift mutation, L1007fs, was detected by Megabace SNuPe genotyping.
Our results show that men who have both the C10X and Q705K alleles in CARD8 and NALP3, and who express wild-type alleles of Nod2 are at an increased risk of developing CD (odds ratio, OR: 3.40 range: 1.32-8.76); P = 0.011). No association with these polymorphisms was found in women (OR: 0.89 (range: 0.44-1.77); P = 0.74).
We suggest a role for combined polymorphisms in CARD8 and NALP3 in the development of CD in men, with obvious sex differences in the genetic susceptibility pattern. These findings give further support to the importance of innate immune responses in CD.
Background
With a lifelong perspective, 12% of ulcerative colitis patients will need a colectomy. Further reconstruction via ileo-rectal anastomosis or pouch can be affected by patients’ perspective ...of their quality of life after surgery.
Aim
To assess the function and quality of life after restorative procedures with either ileo-rectal anastomosis or ileal pouch-anal anastomosis in relation to the inflammatory activity on endoscopy and in biopsies.
Method
A total of 143 UC patients operated with subtotal colectomy and ileo-rectal anastomosis or pouches between 1992 and 2006 at Linköping University Hospital were invited to participate. Those who completed the validated questionnaires (Öresland score, SF-36, Short Health Scale) were offered an endoscopic evaluation including multiple biopsies. Associations between anorectal function and quality of life with type of restorative procedure and severity of endoscopic and histopathologic grading of inflammation were evaluated.
Results
Some 77 (53.9%) eligible patients completed questionnaires, of these 68 (88.3%) underwent endoscopic evaluation after a median follow-up of 12.5 (range 3.5–19.4) years after restorative procedure. Patients with ileo-rectal anastomosis reported better overall Öresland score: median = 3 (IQR 2–5) for ileo-rectal anastomosis (
n
= 38) and 10 (IQR 5–15) for pouch patients (
n
= 39) (
p
< 0.001). Anorectal function (Öresland score) and endoscopic findings (Baron-Ginsberg score) were positively correlated in pouch patients (tau: 0.28,
p
= 0.006).
Conclusion
Patients operated with ileo-rectal anastomosis reported better continence compared to pouches. Minor differences were noted regarding the quality of life. Ileo-rectal anastomosis is a valid option for properly selected ulcerative colitis patients if strict postoperative endoscopic surveillance is carried out.
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