Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we ...demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N:-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.
Citrobacter rodentium induces an acute, self-limited colitis in mice which is histologically associated with crypt hyperplasia. The infection serves as a model for human infectious colitis induced by ...enteropathogenic Escherichia coli. We investigated if Balb/c mice, which had spontaneously cleared C. rodentium infection, were protected against re-infection and if resistance against intestinal infection can be systemically transferred using spleen cells. The course of infection was monitored by faecal excretion. Spleen cells, splenic CD3⁺ and CD4⁺ cells were transferred from resistant mice to non-infected recipients prior to infection. Cytokine secretion, serum and faecal antibody titres and histological disease severity were assessed. Balb/c mice were resistant against re-infection. The course of infection was shorter in mice receiving primed spleen cells, CD3⁺ and CD4⁺ cells. Transfer of CD4⁺ T cells from resistant mice induced γ-interferon, interleukin (IL)-2 and IL-17 secretion and suppressed IL-10 secretion. Anti-Citrobacter serum IgG1 and IgG2a enzyme-linked immunosorbent assay OD levels were increased. Faecal IgA secretion was increased while serum IgA was suppressed in recipients of CD4⁺ cells. Large bowel histology showed protection from colitis in recipients of primed cells as indicated by normal colonic epithelium. In Balb/c mice, C. rodentium infection is followed by resistance, which can be transferred by CD4⁺ cells. Transfer of protection is associated with IL-17 secretion, enhanced serum IgG and faecal IgA secretion. This is the first study to demonstrate the mechanisms by which systemic resistance from previously C. rodentium-infected mice can be transferred to non-infected animals.
Abstract
Background
The efficacy of a single administration of darvadstrocel (expanded allogeneic adipose-derived mesenchymal stem cells) for treating complex perianal fistulas in patients with ...Crohn’s disease was demonstrated in a randomized, double-blind trial (ADMIRE-CD Adipose Derived Mesenchymal Stem Cells for Induction of Remission in Perianal Fistulizing Crohn\'s Disease trial). The current chart review study (INSPECT A retrospectIve chart review study evaluatINg the longer-term effectiveneSs of darvadstrocel in PatiEnts who CompleTed ADMIRE-CD) evaluated the longer-term effectiveness and safety of darvadstrocel.
Methods
Eligible patients had completed at least 52 weeks in the ADMIRE-CD trial. Data on clinical remission and fistula relapse outcomes were collected retrospectively at 104 and 156 weeks after treatment. Adverse events of special interest (tumorigenicity and ectopic tissue formation) were collected up to 208 weeks after treatment.
Results
Eighty-nine patients were included (43 darvadstrocel patients, 46 control subjects). At 52, 104, and 156 weeks posttreatment, clinical remission was observed in 29 (67.4%) of 43, 23 (53.5%) of 43, and 23 (53.5%) of 43 darvadstrocel-treated patients, compared with 24 (52.2%) of 46, 20 (43.5%) of 46, and 21 (45.7%) of 46 control subjects, respectively. In patients with clinical remission at week 52, this remission was sustained at 104 and 156 weeks after treatment in 19 (65.5%) of 29 and 16 (55.2%) of 29 darvadstrocel-treated patients and in 17 (70.8%) of 24 and 13 (54.2%) of 24 control subjects, respectively. Time to fistula relapse and incidence of fistula relapse or new fistula occurrence were not significantly different between groups. Tumorigenicity was reported for 1 (2.2%) patient in the control group (malignant epidermoid carcinoma). No ectopic tissue formation was reported.
Conclusions
Real-world follow-up of patients from the ADMIRE-CD trial indicates that clinical remission of complex perianal fistulas can be sustained in the long term irrespective of whether it is achieved through darvadstrocel administration or maintenance treatment regimens and confirms a favorable long-term safety profile of darvadstrocel.
Lay Summary
This retrospective chart review of patients treated with darvadstrocel indicates sustained remission and confirms a favorable safety profile up to 156 weeks after a single administration of stem cells for treatment of complex perianal fistulas in patients with Crohn’s disease.
SUMMARY
IBD is characterized by increased serum concentrations of different cytokines. IL‐10 inhibits the production of proinflammatory cytokines such as IL‐1, tumour necrosis factor‐alpha (TNF‐a), ...interferon‐gamma (IFN‐γ) and IL‐6 through inhibitory action on Th1 cells and macrophages, and it is thought to be a suppressor type cytokine. In the present study we determined serum concentrations of IL‐10 in patients with ulcerative colitis (UC) and Crohn's disease (CD). We measured human IL‐10 by our own newly established ELISA system using PharMingen antibodies. Serum antibodies were assessed in 44 patients with UC, 40 patients with CD, and in 30 healthy controls. Human IL‐10 serum levels were significantly increased in patients with active UC (144 ± 34 pg/ml (mean ± s.e.m.), P <0.001) and in active CD (132 ± 32 pg/ml, P <0.001) compared with healthy controls (44.9.5pg/ml). Only patients with active CD and active UC presented with significantly increased IL‐10 serum levels, while patients with inactive disease did not show any significant increase. There was no statistically significant difference between IL‐10 serum levels in patients with CD or UC. Compared with clinical disease activity indices there was a significant correlation between IL‐10 serum concentration and CDAI in patients with CD (r= 0.45, P <0.01) and CAI in VC patients (r= 0.39, P <0.05). Comparing IL‐10 serum levels with serum concentrations of other proinflammatory cytokines there was a significant correlation to scrum levels of sIL‐2R (r= 0.417, P <0.05) and IL‐6 (r= 0.387, P <0.05) in patients with CD. Serum cytokine levels in patients with UC did not show any significant correlation to IL‐10 serum concentration. IL‐10 is elevated in serum of patients with active CD and UC. suggesting that IL‐10 acts as a naturally occurring damper in the acute inflammatory process of IBD.
Abstract
Background
Transmural response and healing assessed by intestinal ultrasound (IUS) have gained relevance as outcome measures with the ability to guide treatment decisions in inflammatory ...bowel diseases (IBD). Bowel wall thickness (BWT) is the most widely used IUS parameter and encompasses the thickness of mucosa, muscularis mucosae, and submucosa 1. Recently, the ratio between submucosa/BWT to estimate endoscopic remission was suggested 2 and de Voogd et al. have shown that the submucosa is the most responsive wall layer 3. However, little is known about the association between submucosa hypertrophy and clinical as well as lab parameters.
Methods
TRUST&UC was a prospective, observational study including 224 UC patients with increased BWT at baseline and active disease (SCCAI ≥ 5) with up to 4 visits (baseline, optional visit at week 2 (W2), W6 and W12). At baseline, patients received therapy intensification according to standard of care. In this post-hoc analysis, 171 patients with a W12 visit ± 4 weeks were included. Overall BWT, mucosa and submucosa thickness were evaluated for the sigmoid colon and stratified for SCCAI, CRP, fecal calprotectin (fCal), and Mayo endoscopic subscore (MES).
Results
Active UC patients in our cohort had a median age and disease duration of 37.1 years (28.9 –50.2) and 3.55 years (0.54 – 10.18), more were male (56.6%, n = 95). At baseline, UC patients presented with a high disease activity reflected by a median SCCAI of 9.00 (7.0-10.0). Mean values for BWT, mucosa, and submucosa were 5.54 mm, 1.7 mm, and 2.32 mm, respectively. Over the study course, mucosa and submucosa thickness were significantly reduced with the most pronounced reduction happening within the first 2 weeks (figure 1). Patients with SCCAI ≤ 2 tended to have a more reduced mucosa and submucosa thickness than patients with SCCAI > 2 (fig 2A, A1). Whilst a similar trend was observed for fCal values (not shown), this effect was found neither for CRP (not shown) nor for MES.
Conclusion
We found that both mucosa and submucosa thickness of active UC patients were reduced as a consequence of therapy intensification. The lack of difference in thickness of both mucosa and submucosa in patients with and without MES improvement might indicate fibrotic alterations in these patients which needs further evaluation. Based on these and previous results of our group, we suggest measuring BWT in conjunction with other established IUS parameters such as colour Doppler signal to assess disease activity in UC.
1-Ilvermark et al. JCC 2022
2-Miyoshi et al. J Gastroenterol 2022
3-de Voogd et al. Gastroenterol 2022
Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In ...the following co‐culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll–Hypaque gradient followed by co‐incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco‐2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three‐colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti‐CD4, anti‐CD8, anti‐IFN‐γ and anti‐IL‐4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co‐incubation with Caco‐2 cells we found a significant increase of IFN‐γ‐producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN‐γ+/CD8+ lymphocytes in patients with UC was also seen after direct co‐incubation with primary cultures of colonic crypt cells. The observed epithelial–lymphocyte interaction seems to be MHC I‐restricted. No significant epithelial cell‐mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD—even in an inactive state of disease—exert an increased capacity for IFN‐γ induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN‐γ‐producing CD8+ lymphocytes.
Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and ...chemokines. MCP‐1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL‐4, IL‐10 and IL‐13 have been described to exert anti‐inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP‐1 by activated intestinal epithelial cells. We examined Caco‐2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP‐1 was determined under stimulated and non‐stimulated conditions. IL‐4, IL‐10 and IL‐13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL‐1β or tumour necrosis factor‐alpha (TNF‐α), we observed markedly increased concentrations of MCP‐1 in supernatants of Caco‐2 cells and intestinal epithelial cells. IL‐4, IL‐10 and IL‐13 all had the capacity to down‐regulate the production of MCP‐1 in Caco‐2 cells as well as in freshly isolated epithelial cells. Caco‐2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL‐1β or TNF‐α for MCP‐1 production. As MCP‐1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP‐1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.
It is well known that inflammatory conditions of the intestinal mucosa result in compromised barrier function. Inflammation is characterized by an influx into the mucosa of immune cells that ...influence epithelial function by releasing proinflammatory cytokines such as IFN-gamma and TNF-alpha. Mucosal barrier function is regulated by the epithelial apical junctional complex (AJC) consisting of the tight junction and the adherens junction. Since the AJC regulates barrier function, we analyzed the influence of IFN-gamma and TNF-alpha on its structure/function and determined the contribution of apoptosis to this process using a model intestinal epithelial cell line, T84, and IFN-gamma and TNF-alpha. AJC structure/function was analyzed by confocal microscopy, biochemical analysis, and physiologic measurement of epithelial gate/fence function. Apoptosis was monitored by determining cytokeratin 18 cleavage and caspase-3 activation. IFN-gamma induced time-dependent disruptions in epithelial gate function that were potentiated by coincubation with TNF-alpha. Tight junction fence function was somewhat disrupted. Cytokine treatment was associated with internalization of AJC transmembrane proteins, junction adhesion molecule 1, occludin, and claudin-1/4 with minimal effects on the cytoplasmic plaque protein zonula occludens 1. Detergent solubility profiles of junction adhesion molecule 1 and E-cadherin and their affiliation with "raft-like" membrane microdomains were modified by these cytokines. Inhibition of cytokine-induced apoptosis did not block induced permeability defects; further emphasizing their primary influence on the epithelial AJC structure and barrier function. Our findings for the first time clearly separate the proapoptotic effects of IFN-gamma and TNF-alpha from their abilities to disrupt barrier function.
Helicobacter pylori colonizes the gastric epithelial surface and induces epithelial cells to increase production of the neutrophil attractant IL‐8. Little is known about the role of the gastric ...epithelium in regulating mucosal T cell trafficking. We therefore characterized constitutive and regulated epithelial expression of the CXC chemokines IP‐10, I‐TAC and Mig, which specifically attract CXCR3 expressing CD4+ T cells. Human gastric epithelial cell lines (AGS, Kato III, NCI) were used to characterize the constitutive and regulated expression of three CXC chemokines in response to IFN‐γ, TNF‐α and different H. pylori preparations. Chemokine mRNA and protein production were measured by RT‐PCR and ELISA. Gastric epithelial cells constitutively expressed mRNA for IP‐10, Mig and I‐TAC. IFN‐γ in combination with TNF‐α strongly induced secretion of those chemokines. Soluble or membranous fractions of H. pylori significantly inhibited IFN‐γ/TNF‐α induced epithelial cell IP‐10 and Mig production. Gastric epithelial cells may contribute to mucosal T cell trafficking. The capacity of H. pylori products to inhibit IP‐10 and Mig secretion may explain, at least in part, the failure to induce protective immunity against this bacterium and the ability of H. pylori to affect the presentation of the local inflammation.
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