bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP71 Efficacy, safety, and tolerability of ustekinumab in paediatric patients with moderately to severely active Crohn’s disease: Results from, UniStar, a phase 1 study
Authors: Rosh J , Turner D , Griffiths A , Jacobstein D , Adedokun O , Padgett L , Terry N , O’Brien C , Hyams J
Published: January 2020

Background

Ustekinumab (UST) is approved for the treatment of adults with moderate to severe Crohn’s disease (CD). The objective of this study was to evaluate the pharmacokinetics (PK), safety, and efficacy of UST in paediatric patients with moderately to severely active CD who had failed treatment with corticosteroids (CS) and/or immunomodulators (IM) and/or anti-tumour necrosis factor (TNF) therapies. Here, we report the safety and efficacy results through Week 16; PK results are reported separately.

Methods

This was a Phase 1, multicentre, 16-week, double-blind induction dose-ranging study (NCT02968108) in patients aged 2 to <18 years (body weight [BW] ≥10 kg) with a Paediatric CD Activity Index (PCDAI) score >30 and at least an abnormal C-reactive protein >3.0 mg/l or faecal calprotectin >250 µg/g), or ulcerations in the ileum or colon upon ileocolonoscopy despite adequate treatment with CS ± IM ± anti-TNF therapy. Patients were randomised (1:1) and stratified by weight and prior anti-TNF use for induction to one of 2 weight range-based intravenous (IV) doses: 130mg vs. 390 mg if BW ≥40 kg and 3 mg/kg vs. 9 mg/kg if BW <40 kg. At week 8, all patients received a single subcutaneous (SC) UST maintenance dose of 90mg if BW ≥40 kg or 2 mg/kg if BW <40 kg.

Results

Forty-four patients (59% ≥40kg BW; >90% anti-TNF exposed) were randomised (n = 23 lower dose; n = 21 higher dose) and treated with UST. Baseline characteristics are summarised in Table 1. At week 16, in the lower dose and higher dose groups, 52%/52% achieved clinical response (reduction in PCDAI ≥15) and 22%/29% had clinical remission (PCDAI ≤10), respectively (Table 2). In addition, 32% and 28% of patients showed endoscopic response (reduction in Simple Endoscopic Score for CD of ≥50%), respectively. Through week 16, 73% of patients reported ≥1 adverse event (AE; 82.6% lower dose vs. 62% higher dose); 2 discontinued due to AEs (1 in each group). Serious AEs occurred in 16% of patients (26% lower dose and 5% higher dose, with CD exacerbation being the most frequent (13%/5%, respectively). Infections occurred in 41% of patients (1 was serious, which was intestinal abscess that spontaneously resolved with sequelae). No injection site reactions, opportunistic infections, malignancies, or deaths were reported. No antibodies to UST were observed.

Conclusion

As early as 3 weeks and through 16 weeks, both the lower and higher doses of UST (IV week 0 and SC at week 8) improved clinical and endoscopic disease activity in this previously treatment-refractory group of children with CD. The safety profile was consistent with that for UST in adults with CD.
DOP83 Intestinal fibrosis in Crohn’s disease patients is marked by up-regulation of innate immune cells and mucosal B cells
Authors: Creyns B , Gabriele D , Jonathan C , Verstockt B , Bislenghi G , Marc F , Vermeire S , Van Assche G , Ceuppens J , D’Hoore A , De Hertogh G , Breynaert C
Published: January 2020

Background

Intestinal fibrosis represents a significant complication of inflammatory bowel disease (IBD). An urgent need is present for the identification of pathways and markers involved in fibrogenesis to prevent and intervene with intestinal fibrosis. As intestinal fibrosis mainly occurs in the intestinal deeper layers, mucosal biopsies for studies in intestinal fibrosis are of limited information. Therefore, characterisation of the relative contribution of innate and adaptive immune cells in the mucosa and deeper layers in fibrotic distal ileum of Crohn’s disease (CD) patients was performed.

Methods

Seventeen CD patients undergoing right hemicolectomy (RHC) for stricturing disease and 6 colorectal cancer (CRC) patients undergoing RHC were recruited. The resected ileum was divided in macroscopically inflamed and fibrotic tissue and single-cell suspensions were made from mucosal and deeper intestinal layers for immune cell characterisation with flow cytometry. For comparison, proximal unaffected CD tissue and control ileum from CRC patients was included. Fibrosis and inflammation were confirmed on HE stained histological sections. Wilcoxon matched-pairs signed-rank test between CD samples and Kruskal–Wallis testing with Dunn’s multiple comparisons test compared with CRC ileum were performed.

Results

From 12 CD patients, an additional macroscopically inflamed region could be identified next to the fibrotic area, with a decreased fibrosis score as compared with the fibrotic area (4.00 vs. 6.00, p = 0.016) (Figure 1). Both fibrotic and inflamed regions had increased inflammation as compared with proximal unaffected CD and control non-IBD ileum (score: 4.00 and 4.50 vs. 1.00 and 0.00, p < 0.001 for all). In the inflamed ileum, no differences in immune populations were observed between mucosa and deeper layers, reflecting the transmural nature of CD. In contrast, CD19+ B cells were specifically enriched in the mucosa of fibrotic ileum, as compared with proximal CD mucosa (32.20 vs. 20.40% of CD45+ cells, p = 0.008) (Figure 2). In the deeper layers of fibrostenotic CD ileum FcεR+ Siglec 8+ eosinophils (1.13 vs. 1.17 % of CD45+ cells, p = 0.027), mature CD11c+ dendritic cells (3.95 vs. 2.96 % of CD45+ cells, p = 0.042) and M2 CD206+ macrophages (0.35 vs. 0.18% of CD45+ cells, p < 0.001) were enriched as compared with the mucosa overlying the fibrotic tissue (Figure 3).

Conclusion

These results argue that inflammation in the deeper intestinal layers is different from the inflammatory signature seen in mucosal inflamed regions. We here report alternative innate immune cells expanded specifically in the deeper intestinal layers of fibrostenotic CD ileum that could identify targets for new anti-fibrotic therapies.
DOP84 Crohn’s disease modifies the DNA methylome of human adipose-stem cells, which is only partially re-established in remission
Authors: Serena C , Millan M , Ejarque M , Saera-Vila A , Monfort-Ferré D , Bautista M , Menacho M , Martí M , Espin E , Vendrell J , Fernández-Veledo S
Published: January 2020

Background

Crohn’s disease (CD) is characterised by the expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-tissue stem cells (hASCs) from CF exhibit a dysfunctional phenotype, including a pro-inflammatory profile, high phagocytic capacity and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission, and are found in all adipose depots explored including subcutaneous fat. We hypothesised that this is a consequence of epigenetic modifications.

Methods

Epigenome-wide association studies using the 850k EPIC Illumina array were used to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat (Figure 1). Changes in the expression of differentially methylated candidate genes were validated in hASCs of patients with active/inactive CD and healthy controls (Figure 1).

Conclusion

hASCs of patients with CD exhibit an alter DNA-methylation and gene expression profile. Remarkably, the expression of several genes is only partially restored in patients with inactive CD. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.
P010 Measurement of colonic mucosal content of biologics with the use of magnetic resonance imaging (MRI): A pilot study in patients with ulcerative colitis
Authors: Filip R , Bar P , Bartusik-Aebisher D , Aebisher D
Published: January 2020

Background

Anti-tumour necrosis factor antagonists (infliximab) as well as other molecules with different modes of action, including anti-integrin agents (vedolizumab), are currently used in patients with ulcerative colitis (UC Numerous studies have demonstrated a positive correlation between serum biologic drug concentrations and favourable therapeutic outcomes, whereas low or undetectable drug concentrations can lead to treatment failure. However, despite immunological issues, lack of and or loss of response may also be attributed to drug pharmacokinetics, of which penetration to the target tissue (colon wall) may play a crucial role

Methods

We used MRI to perform biochemical analyses of infliximab, adalimumab and vedolizumab concentrations testing the hypothesis that MRI relaxation time can be used to track antibodies in both mucosal biopsy samples and serum. All MR scans were performed with an Optima MR360 from General Electric Healthcare. To determine spin–lattice (T1) and spin–spin (T2) relaxation times, the Fast Spin Echo (FSE) sequence was used.

Results

The measured values of T1 relaxation times for infliximab, adalimumab, and vedolizumab were 2227 ± 35 ms, 2000 ± 22 ms and 1288 ± 15 ms, respectively. The obtained T2 relaxation times were 130 ± 11 ms, 90 ± 5, and 75 ± 10 ms, respectively. A decrease of both T1 and T2 values of 15 ± 3% are observed in serum from patients with ulcerative colitis. The values of infliximab and adalimumab were similar; the values of vedolizumab measurements in serum were about 50% lower. We find primary evidence that in T1 and T2 decreased in serum samples with ulcerative colitis and increase with the administration of infliximab, adalimumab and vedolizumab drugs. Samples of healthy tissue have T1 and T2 in the range of 2700 ± 5 ms and 150 ms ± 5 ms, respectively. A 30% decrease in T1 and T2 are observed for samples with ulcerative colitis. In this pilot study, we observed that values of T1 and T2 for tissues and serum that contain infliximab and adalimumab are similar, but vedolizumab shows a difference of about 30% when compared with infliximab and adalimumab.

Conclusion

MRI is an excellent method for quantitative and qualitative measurements of drug content in tissues and biological fluids. This is an innovative use of magnetic resonance imaging to develop a methodology for imaging of drugs that act as contrast agents via interaction with water in serum and tissue.
P038 A single-cell transcriptomics approach reveals high-resolution cellular signatures in Crohn’s disease
Authors: Garrido Trigo A , Corraliza A M , Veny M , Lozano J , Ricart E , Ordás I , Masamunt M C , Panés J , Hemberg M , Salas A
Published: January 2020

Background

Single-cell RNA sequencing (sc-RNAseq) provides high-resolution analysis of individual cells and cell clusters within complex tissues like the intestinal mucosa. No study to date has applied this novel technology to understand the complexity of intestinal inflammation in Crohn’s disease (CD) patients. Our aim is to identify the cellular subsets present in healthy and inflamed colon, and to describe how individual subsets are regulated in active CD.

Methods

Colon biopsies from a healthy control (HC) and two patients with endoscopically active colonic CD were collected and processed by enzymatic digestion and cell sorting to discard dead cells. Single-cell suspensions were processed by droplet-based protocol 10× chromium system followed by library preparation and sequencing. Sequencing data were aligned and quantified using Cell Ranger. Downstream analysis was performed using the R package Seurat.

Results

scRNA-seq analysis identified 14 transcriptionally different cell subsets in the healthy colon, including diverse types of T cells, plasma cells, mesenchymal cells, epithelial cells and myeloid populations, as well as a subset of MHCII+ CD20+ B cells. Overall, the number of transcriptionally differentiated cellular subsets was greater (16 and 17 subsets per sample) in the two CD colonic samples revealing higher cellular heterogeneity in active CD. In particular, in the HC we identified three T-cell subsets including a CD4+, CD8+ and a smaller subset of intraepithelial lymphocytes. In contrast, samples from CD patients presented four to six clearly differentiated T-cell clusters depending on the patient, with a marked enrichment of cytotoxicity genes (GZMA, GZMB, NKG7, CCL5 and AOAH) and T-regulatory genes (FOXP3, CTLA4, BATF and IL2RA). In addition, the HC sample presented three differentiated subsets of antibody-secreting plasma cells with predominant IgA and IgM production. In contrast, a clear expansion of IgG-producing plasma cells was observed in both CD patients. Moreover, the profiles of specific subsets significantly changed revealing transcriptional regulation within the individual subsets. Specifically, macrophages within the HC mucosa expressed the M2 markers CD163 and CD209 together with a whole signature of 163 additional genes, while in CD, they lost expression of these markers and upregulated CXCL8 and IL1B which were co-expressed with 268 additional genes common to macrophage populations in both CD patients.

Conclusion

We show that sc-RNAseq can be applied to study at a high cellular resolution human intestinal biopsies. Understanding the transcriptomic profile of the CD-related subsets will help dissect disease pathophysiology and provide knowledge for therapeutic targets.
P043 Small intestinal inflammation but not colitis drives pro-inflammatory nutritional antigen-specific T-cell response
Authors: Rodríguez Sillke Y , Schumann M , Lissner D , Branchi F , Glauben R , Siegmund B
Published: January 2020

Background

Inflammatory bowel disease (IBD) represents a dysregulation of the mucosal immune system. The pathogenesis of Crohn’s disease (CD) and ulcerative colitis (UC) is linked to the loss of intestinal tolerance and barrier function. The healthy mucosal immune system has previously been shown to be inert against food antigens. Since the small intestine is the main contact surface for antigens and therefore the immunological response, the present study served to analyse food-antigen-specific T cells in the peripheral blood of IBD patients.

Methods

Peripheral blood mononuclear cells of CD, with an affected small intestine, and UC (colitis) patients, either active or in remission, were stimulated with the following food antigens: gluten, soybean, peanut and ovalbumin. Healthy controls and celiac disease patients were included as controls. Antigen-activated CD4+ T cells in the peripheral blood were analysed by a magnetic enrichment of CD154+ effector T cells and a cytometric antigen-reactive T-cell analysis (‘ARTE’ technology) followed by characterisation of the effector response.

Results

The effector T-cell response of antigen-specific T cells were compared between CD with small intestinal inflammation and UC where inflammation was restricted to the colon. Among all tested food antigens, the highest frequency of antigen-specific T cells (CD4+CD154+) was found for gluten. Celiac disease patients were included as control, since gluten has been identified as the disease-causing antigen. The highest frequency of gluten antigen-specific T cells was revealed in active CD when compared with UC, celiac disease on a gluten-free diet (GFD) and healthy controls. Ovalbumin-specific T cells were almost undetectable, whereas the reaction to soybean and peanut was slightly higher. But again, the strongest reaction was observed in CD with small intestinal involvement compared with UC. Remarkably, in celiac disease on a GFD only antigen-specific cells for gluten were detected. These gluten-specific T cells were characterised by up-regulation of the pro-inflammatory cytokines IFN-γ, IL-17A and TNF-α. IFN-g was exclusively elevated in CD patients with active disease. Gluten-specific T-cells expressing IL-17A were increased in all IBD patients. Furthermore, T cells of CD patients, independent of disease activity, revealed a high expression of the pro-inflammatory cytokine TNF-α.

Conclusion

The ‘ARTE’-technique allows to analyse and quantify food antigen specific T cells in the peripheral blood of IBD patients indicating a potential therapeutic insight. These data provide evidence that small intestinal inflammation in CD is key for the development of a systemic pro-inflammatory effector T-cell response driven by food antigens.
P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease
Authors: Gisbert-Ferrandiz L , Cosín-Roger J , Coll S , Bauset C , Ortiz-Masiá D , Navarro-Vicente F , Alos R , Calatayud S , Barrachina M D
Published: January 2020

Background

Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts.

Methods

We used intestinal fibroblasts isolated from surgical resections of the damaged mucosa of CD patients with stricturing behaviour (B2). Control fibroblasts were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with VD (100 nM) or its vehicle for 24 h and PDIA3 protein levels were measured by Western Blot. In the wound healing analysis, a single scraping was done in the centre of the fibroblasts monolayer and FBS-free medium was added to the cells, which allows us to determine the ability of fibroblasts to migrate and close the wound. Photos were taken at 0, 24 and 48 h. Results of wound healing were expressed as the percentage of the wound at each time point for the maximal wounded area (time 0, 100%). Statistical significance was measured by ANOVA or t-test.

Results

No significant differences in PDIA3 protein levels were detected between control and CD fibroblasts but VD significantly decreased PDIA3 expression in CD fibroblasts (Figure 1A). In the wound healing assay, we detected that CD-B2 fibroblasts migrate faster than control cells, resulting in a reduced wounding area, 48 h later (Figure 1B). Treatment of these CD-B2 cells with VD decreased their migration rate, and 48 h later cells exhibited a higher and significant wounding area than vehicle-treated cells (Figure 1C).

Conclusion

Vitamin D decreased PDIA3 expression and prevented the accelerated migration detected in intestinal fibroblasts from B2-CD patients which suggest an anti-fibrotic effect of VD mediated by a direct effect of this hormone on intestinal fibroblasts.
P054 The impact of intestinal stenosis on the expression of Crohn’s disease in young mice with genetic predisposition
Authors: Georgopoulos I , Mavrigiannaki E , Stasinopoulou S , Renieris G , NIkolakis G , Kollias G , Tiniakos D , Papaconstantinou I
Published: January 2020

Background

In the majority of patients, the areas of the intestine affected in Crohn’s disease (CD) are the terminal ileum and less commonly the rectum. The terminal ileum is affected in the majority of genetic animal models of CD as well. Additionally, CD recurs at sites of tight anastomosis or strictures postoperatively. These observations lead to the assumption that increased intraluminal pressure related to the presence of a valve, sphincter or stenosis is associated with CD pathogenesis. We investigated the hypothesis that the creation of a partial intestinal stenosis could have an impact on disease severity, in a genetic animal model of CD (TNFΔare/+). TNFΔare/+ mice overexpress TNFα leading to a Crohn-like colitis in the terminal ileum.

Methods

Twenty-nine TNFΔare/+ mice, 6 weeks old, were divided into three intervention groups: stenosis, sham and control. In the stenosis group (n = 11), a partial small bowel obstruction was created via a novel triple suture technique, approximately 3 cm from the ileocecal valve. In the control group (n = 9), a loose single suture was placed at the aforementioned site to test the direct effect of the foreign material on the intestinal wall. The sham group (n = 9) received a sham operation. The triple suture stenosis was also performed on wild-type (WT) C57BL/6 mice (group WT, n = 9). 6 weeks post-surgery all animals were sacrificed and samples from the ileum 3 cm proximal and 3 cm distal to the intervention site were collected for histopathological evaluation. The Crohn-like changes were assessed using a modified colitis histological scoring system (based on Katakura et al., JCI 2005; 115: 695–702).

Results

Proximal to the intervention, the mean colitis score of stenosis group (10.18 ± 0.87) was significantly higher compared with sham (6.33 ± 0.97, p: 0.009) and control group (5.00 ± 0.91, p: 0.001). There was no difference between sham and control group (p: 0.332). No significant differences between the groups were reported distal to the intervention. The triple suture technique led to Crohn-like inflammatory lesions only in the TNFΔare/+ mice, as shown from the significantly increased score compared with WT mice proximal and distal to the stenosis (10.18 ± 0.87 vs. 0.67 ± 0.37, p < 0.001 and 9.20 ± 1.09 vs. 0.33 ± 0.24, p < 0.001).

Conclusion

The creation of a stenotic segment in the intestine of TNFΔare/+ mice, led to higher colitis score than expected. The probable mechanism is the increased intraluminal pressure proximal to the stenosis. This suggests that mechanical forces contribute as important co-factors in the pathophysiology of CD, in genetically predisposed populations.
P060 The pharmacologic inhibition of store-operated calcium entry pathway in inflammatory bowel disease
Authors: Letizia M , Yerinde C , Sand A , Schlickeiser S , Kaufmann U , Siegmund B , Feske S , Weidinger C
Published: January 2020

Background

Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients.

Methods

PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition.

Results

Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells.

Conclusion

Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.
P064 Exosomes from mesenchymal stromal cells reduce murine colonic inflammation via a macrophage-dependent mechanism
Authors: Wu X R , Lan P , Wu X J , Gao X
Published: January 2020

Background

Conventional treatments for inflammatory bowel disease (IBD) have multiple potential side effects. Therefore, alternative treatments are desperately needed. Exosomes from mesenchymal stromal cells (MSC-Exos) show potent immunomodulatory activities and protective effects in several diseases.

Methods

MSC-Exos were isolated from human bone marrow-derived mesenchymal stromal cells. Experimental colitis was induced by administration of dextran sulphate sodium (DSS) in C57BL/6 mice (male, 6–8 weeks), or 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) in Balb/c mice (male, 7–9 weeks). Macrophage depletion was performed via intraperitoneal injection of clodronate liposomes. Colitic mice were treated intravenously with MSC-Exos or saline, and mortality and diverse disease signs were tracked.

Results

Systemic administration of MSC-Exos significantly mitigated colitis in mice. MSC-Exos treatment downregulated inflammatory responses, maintained intestinal barrier integrity and polarised M2 macrophages. Infused MSC-Exos mainly acted on colonic macrophages and their beneficial effect was blocked by macrophage depletion. Particularly, MSC-Exos were enriched in proteins involved in regulating multiple biological processes associated with the anti-colitic benefit of MSC-Exos.

Conclusion

MSC-Exos are critical regulators of immune/inflammatory responses and may be promising candidates for IBD treatment
P066 Azathioprine linked with impaired intestinal epithelial regeneration in Crohn’s disease
Authors: Borycka-Kiciak K , Pietrzak A , Ferenc K , Pietrzak P , Janaszek L , Tarnowski W , Zabielski R
Published: January 2020

Background

In patients operated due to active Crohn’s disease (CD), the negative effect of steroids and biologics on septic complications is known. Until now, azathioprine (AZA) is considered to be safe. The aim of our study was to assess the impact of AZA on intestinal epithelial cells damage in patients with active CD as a surrogate marker of healing.

Methods

Intestinal specimens taken from macroscopically healthy surgical margins of all consecutive CD patients operated due to active isolated ileocecal disease during the study period (2014–2016) were evaluated. Expression of caspase-3, p-53 and Ki-67 as markers of cell apoptosis, DNA damage and proliferation were immunohistochemically tested and assessed using a confocal microscope and microimage for in-tissue-cytometry analysis. Western-blot analysis was performed for the evaluation of cellular integrity using ZO-1 and E-cadherin as a markers. 30-day clinical outcomes were assessed. The study was approved by the institutional Ethics Committee.

Results

From 61 operated due to active CD patients, 35 met the inclusion criteria. Patients were divided accordingly to preoperative treatment: treated with no immunomodulators (N-9 patients), on steroids (S-14 patients), on AZA (A-6 patients), and on combination therapy, AZA + steroids (AS-6 patients). There were no substantial differences between groups. We found statistically significant increase of apoptosis in A group compared with N (5.33 ± 1.05 vs. 1.29 ± 0.51, p = 0.011), but also S group (1.58 ± 0.68, p = 0.014) and increase of DNA damage in A, AS and S groups compared with N group (p =0.01; p = 0.032; p = 0.035, respectively) (Figure 1a–c). P53-mediated cell cycle arrest and apoptosis through a caspase-3-dependent pathway in response to DNA damage was the most intensive in A group (Figure 2a). Reduction of cell proliferative activity in group A did not reach statistical significance (p = 0,057). A reduction of ZO-1 in A group and increased level of E-cadherin in S group were found. The effect of the decreased number of tight junctions and disintegration of the mucosa layer was observed in A group (Figure 2b). Clinically, in 30-day postoperative follow-up, six wound healing complications and one anastomotic leak were found, all in patients treated with immunomodulators.

Conclusion

We found that in epithelial cells of the small and large intestine of patients treated with AZA, apoptotic activity and DNA damage processes are increased when regeneration processes and mucosal integrity are significantly disturbed. These abnormalities of intestinal epithelial regeneration may be a surrogate marker of impaired mucosal healing.
P084 Cannabis use promotes lymphocyte responsiveness and modifies T-cell signalling in adolescent IBD patients
Authors: Sanctuary M , Wilkinson C , Jones A , Murphy B , Hoffenberg E , Collins C
Published: January 2020

Background

The prevalence of inflammatory bowel disease (IBD) has increased dramatically in recent years, particularly in paediatric populations. The efficacy of available therapies is limited and often transient, leading patients to seek alternative therapies for symptom relief, including the use of medical marijuana (Cannabis sativa). There is some evidence to suggest that cannabinoids exert anti-inflammatory effects in preclinical colitis models. In addition, a number of small studies have reported improvements in non-empirical disease measures and reductions in visceral pain in response to cannabis use. However, associations between cannabis use in IBD and increased need for surgical interventions have also been reported.

Methods

Therefore, determining the direct impact of cannabis use on immune modulation in IBD patients is of critical importance. Blood samples collected from paediatric IBD patients who reported cannabis use for symptom control were analysed for cytokine expression and T-cell signalling pathway activation compared with non-users. Concentrations of serum phytocannabinoids were determined by HPLC and correlated with subsequent ex vivo assay parameters.

Results

Results demonstrated elevated levels of a myriad of pro-inflammatory cytokines in users vs. non-users upon ex vivo restimulation. This coincided with an expansion of pathogenic Th17 cells in the periphery. Differences in signalling cascades of activated T cells between users and non-users were also observed.

Conclusion

These results suggest that cannabis exposure, which can desensitise cannabinoid receptors, may prime pro-inflammatory pathways in paediatric IBD patients. Future studies should address the limitations of observational studies through the use of randomised controlled trials of cannabis use in paediatric IBD populations.

This research was funded by the Colorado Department of Public Health and Environment.
P090 Influence of high-fat diet on colitis and liver health in inflammatory bowel disease: a study using murine model of DSS-induced colitis
Authors: Choe A R , Kim S E , Lee H A , Yoo S G , Kim T O , Tae C H , Moon C M , Jung H K , Shim K N , Jung S A
Published: January 2020

Background

In recent, consumption of high-fat (HF) diet has been increasingly considered to be a significant aggravating factor of inflammatory bowel disease and non-alcoholic steatohepatitis. The aim of this study was to investigate whether exacerbation of chronic DSS-induced colitis caused by HF diet could promote liver inflammation, using measurement of biomarkers.

Methods

Male C57BL/6 mice were allocated into four experimental groups receiving either; (1) control with normal diet/water; control, (2) control with high-fat diet; HF, (3) DSS induced colitis group with normal diet/water; DSS, (4) DSS-induced colitis group with high-fat diet; DSS + HF for 4 weeks. On day 30 of study, colon and liver tissues samples were obtained for measuring mRNA expression of pro-inflammatory/pro-fibrogenic markers and blood for metabolic parameter analysis. Spearman rank correlations were analysed between colon and liver inflammatory biomarkers.

Results

The mean colon length was shorter, and more stenotic lesions were observed in DSS + HF group, compared with the other three groups. While HF diet alone did not cause significant colonic inflammation, HF diet in chronic DSS colitis aggravated colonic inflammation revealing the significant increase of IL1-β and IL17 mRNA expression as well as increasing the disease activity indices significantly at the late phase of the study. Moreover, DSS + HF group showed significantly higher serum levels of total cholesterol (p = 0.004) and glucose (p = 0.002), compared with HF group. Hepatic inflammation was more severe in DSS + HF group, showing significantly increased IL1-β and TGF-β expression compared with HF group. The mRNA expression of IL-1β in colitis group was statistically positive correlated with those in liver tissue (rs = 0.51, p = 0.010).

Conclusion

High-fat diet in chronic colitis may adversely affect the metabolic parameters and liver inflammation, as well as aggravation of colitis.
P091 Dietary triggers of colonic inflammation following treatment with exclusive enteral nutrition in children with Crohn’s disease
Authors: Gkikas K , Logan M , Nichols B , Clark C M , Ijaz U Z , Gervais L , Duncan H , Garrick V , Curtis L , Buchanan E , Cardigan T , Armstrong L , Delahunty C , Flynn D M , Barclay A R , Tayler R , Hansen R , Russell R K , Gerasimidis K
Published: January 2020

Background

Exclusive enteral nutrition (EEN) ameliorates gut inflammation in children with Crohn’s disease (CD). We have previously described the rapid rise in faecal calprotectin levels (FC) when children with CD return to their habitual diet after EEN treatment (1). We performed dietary assessment and measured biomarkers of food consumption in faeces of children with CD during early food reintroduction.

Methods

A composite outcome of clinical remission post EEN (weighted Paediatric Crohn’s Disease Activity Index <12.5) and a significant drop in FC (FC decrease: >500 mg/kg / >35%) was used to define the patient group. All patients completed 3-day estimated weight food diaries and provided a faecal sample. Patients were divided equally for statistical analysis purposes in two groups; above (Group 1) and below (Group 2) the median FC concentration at food reintroduction. Nutrient analysis was performed with WinDiets. Short-chain fatty acids, the gluten immunogenic peptide (GIP) and starch were measured in stool, as proxy of fibre, gluten and malabsorbed starch, respectively. Food groups were assigned using the National Diet and Nutrition Survey approach and statistics employing taxonomy were utilised in the food group analysis. All data are displayed using medians (Q1, Q3).

Results

Fourteen children provided an FC sample within 21 (15, 51) days post EEN. Patients were of similar age, weight and height (p = 0.4, p = 0.37, p = 0.79). Total energy intake did not differ between the groups (p = 0.37). Patients in Group 1 consumed more fibre than Group 2 [12.1 g (11.3, 19.9) vs. 9.9 g (7.6, 12.1), p = 0.04]. Protein and phosphorus intakes, expressed as a percentage of reference nutrient intakes, were also higher in Group 1 than Group 2 [protein (%): 262 (195, 291) vs. 211 (88, 215), p = 0.02], [phosphorus (%): 241 (171, 290) vs. 177 (131, 1901), p = 0.04]. Butyrate and valerate levels were higher in Group 1 than Group 2 [butyrate: 62.5 (41.8, 114) vs. 34.1 (29.2, 49.6), p = 0.02; valerate: 13.7 (9.3, 17.9) vs. 4.5 (2.3, 8.1), p < 0.01. There were no differences in GIP and faecal starch levels between the two groups. Red and processed meat intake was higher in Group 1 than Group 2 [151 g (66.7, 190) vs. 63.3 g (21.7, 67), p = 0.02)]. Cereals intake was non-significantly higher in Group 1 [389 (207, 405) vs. 231 (141, 279), p = 0.08]. Overall diet diversity did not differ between the two groups (p = 0.47).

Conclusion

The current analysis suggests that fibre, protein, phosphorus and red and processed meat may be associated with recurrence of colonic inflammation in children with CD during early food reintroduction. These findings should be confirmed in larger studies.

Reference
P093 Crosstalk between Toll-like receptor 4 and enteric serotonergic pathways in a mouse model of dinitrobenzene sulfonic acid-induced colitis
Authors: Cerantola S , Ridolfi M , Faggin S , Tegon M , Bussolaro G , Salviato E , Bistoletti M , Caputi V , Giaroni C , Giron M C
Published: January 2020

Background

Changes in serotonin (5-HT) levels, anomalies in serotonergic and cholinergic machinery and altered Toll-like receptor 4 (TLR4) expression have been shown in IBD in patients and related animal models. Thus, we aimed to assess the crosstalk of enteric serotonergic system and TLR4 signalling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced colitis.

Methods

Male C57/Bl6 (WT) and TLR4−/− mice (9 ± 2 weeks old; N = 10 mice) were presensitised with 1% dinitrobenzene sulfonic acid (DNBS), and after 1 week was intrarectally instilled with 2.5% DNBS. Small intestine inflammation was measured by disease activity index and histological analysis. Changes in ileal muscle tension were isometrically recorded following: (1) cumulative addition of carbachol (CCh; 0.1–100 µM); (2) electric field stimulation (EFS, 0–40 Hz); (3) 60 mM KCl; (4) 30 μM 5-HT addition with or without 0.1 μM ondansetron (5-HT3R antagonist). Immunofluorescence distribution of the neuronal HuC/D and nNOS and glial GFAP markers were determined in longitudinal-muscle-myenteric plexus whole mounts (LMMPs) by confocal microscopy.

Results

In WT mice, DNBS treatment altered receptor and not-receptor mediated responses (+120% of Emax to CCh and +103% of contraction to KCl, respectively; p < 0.001, N = 5 mice/group) together with an altered cholinergic neurotransmission (−50% at 10 Hz; p < 0.01, N = 5 mice/group) and 2-fold increase to 30 μM 5-HT-mediated response (p < 0.001, N = 5 mice/group). After DNBS treatment TLR4−/− mice showed a significant increase in excitatory-mediated response (+98% of Emax to CCh; +80% of contraction to KCl; +120% at 10 Hz; p < 0.001, N = 5 mice/group) together with a significant reduction of 30 μM 5-HT-mediated response (−50%, p < 0.001, N = 5 mice/group). These changes were associated to a significant decrease of the total number of HuC/D+ neurons (−44% and −19% for WT DNBS and TLR4 DNBS mice, respectively) together with a 1.3-fold increase in S100b immunofluorescence in WT mice after DNBS treatment.

Conclusion

These findings not only suggest an important role of TLR4 in small intestine neuromuscular dysfunction during colitis but also provide novel information on the potential benefits of targeting TLR4 in various gut disorders that exhibit aberrant cholinergic and 5-HT signalling.
P098 Receptor-interacting protein 1 kinase inhibition therapeutically ameliorates experimental T-cell-dependent colitis in mice
Authors: Gobbetti T , Kathryn F , Beal A , Rowles A , Pearse G , Harada I , Bertin J , Haynes A , Berger S
Published: January 2020

Background

Inflammatory bowel disease (IBD) is a multifactorial disorder characterised by chronic and relapsing intestinal inflammation. Receptor-interacting protein 1 (RIP1) kinase activity is emerging as a driver of pro-inflammatory cytokine production and cell death and has been implicated in multiple inflammatory diseases including IBD. To this end, recent work has shown that RIP1 kinase inhibition reduces the spontaneous production of cytokines from human ulcerative colitis (UC) and Crohn’s disease explants.

Methods

The aim of this study was to investigate the anti-inflammatory effect of a highly selective inhibitor of RIP1 kinase activity (GSK547A) in the mouse T-cell transfer model of colitis; a model that shares many features with human IBD. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Chronic colitis was achieved by transferring CD4+CD45RBhigh T cells into immunodeficient female SCID mice (6/8 weeks old). After confirming the development of pathology using endoscopy and MRI, animals were treated therapeutically with the RIP1 inhibitor GSK547A (50 mg/kg twice a day PO) or vehicle (0.5% hydroxypropyl methylcellulose in water). The severity of colitis was measured 5 weeks after cell transfer both macro- and microscopically. Mucosal damage by endoscopy was also assessed. RT-PCR and MSD analysis were performed to detect colon cytokine/chemokine and calprotectin levels. SAA in the plasma was measured using ELISA.

Results

We found that treatment with GSK547 significantly ameliorated experimental T-cell-dependent colitis in mice. GSK547A-treated mice displayed decreased weight loss, colon density (ratio weight/length), macroscopic disease activity index, colon thickness compared with vehicle-treated animals. Mucosal damage, assessed by endoscopy and histopathology, was also reduced following treatment with RIP1 kinase inhibitor. In addition, GSK547A reduced the expression of cytokines in the colon (TNF-α, IL-17A, INF-γ, IL-6 and MCP-1), both at a protein and RNA level. Relevant translational biomarkers such as SAA in plasma and RNA calprotectin in the colon were also decreased in the GSK547A treated group when compared with vehicle.

Conclusion

Our results suggest that RIP1K inhibition is a strong protective factor with anti-inflammatory potential in the progression of chronic colitis, when applied to the translationally relevant T-cell transfer model of colitis. These findings suggest the potential application in the management of inflammatory bowel disease and support the ongoing clinical program evaluating the effect of RIP1 kinase inhibition in UC patients.
P104 Defective metabolism mediates NK cell dysfunction in inflammatory bowel disease (IBD)
Authors: Zaiatz Bittencourt V , Ryan E , Jones F , Doherty G
Published: January 2020

Background

Cell metabolism, in addition to providing energy and biochemical building blocks, also regulates immune cell function. We have increased our understanding of how natural killer cells (NK cell) metabolism impacts human NK cell function; however, little is known about NK cell role and metabolic changes in autoimmune diseases. The dysfunctional NK cell phenotype observed in pathological settings linked to defective cell metabolism will give insights into the development of new therapies for inflammatory diseases. IBD is an autoimmune disease of unknown origin characterised by severe gut inflammation and increased risk of colorectal cancer. NK cells can augment autoimmune responses through their activation of adaptive immune response by interaction with macrophages, dendritic cells and T cells; therefore, it is essential to decipher NK cells function in IBD to propose novel therapeutic targets. In this study, we defined the phenotype and metabolic profile of NK cells isolated from peripheral blood of IBD patients.

Methods

Blood samples were obtained from healthy donors and IBD patients from whom written consent had been obtained. PBMC were isolated by Lymphoprep (Axis-Shield) gradient. PBMC were stained with a combination of cell surface receptors, intracellular cytokines, proteins and analysed by flow cytometry. Cells were either stained ex vivo or after overnight stimulation with cytokine combination IL-12 + IL-15 and in the presence or absence of mTORC1 inhibitor rapamycin. Changes in OxPhos or glycolysis were detected by XFp extracellular ﬂux analyser (Agilent Technologies). Mitochondria mass and polarisation were measured by flow cytometry utilising MitoTracker Green and MitoTracker Red, respectively (ThermoFisher).

Results

NK cells from IBD patients have impaired mTORC1 activity (a key signalling pathway regulating cell metabolism), which is associated with increased expression of pro-inflammatory cytokines IL-17 and TNF-α. Patients NK cells have dysfunctional mitochondria characterised by reduced mitochondria mass, low membrane potential and limited OXPHOS and glycolysis. Furthermore, patients NK cells have limited IFNγ production, reduced TRAIL expression and defective capability of killing cancer cells.

Conclusion

Taken together, our results strongly imply that NK cells from IBD patients have a distinct bioenergetic programme that favours the increased production of pro-inflammatory cytokines. This might be an important unknown defect of NK cells in the regulation of inflammation in IBD that can be used for the development of new treatments. Further studies need to be conducted to identify the reason for the observed defects and determination of other key metabolic pathways involved in NK-cell function during IBD.
P107 The expression and regulation of Oncostatin M and its receptor in intestinal inflammation
Authors: Cineus R , Boesel D , Hainbuch S , Jukes C , Hsieh Y H , Reich J , Borek Z , Kuehl A , Hegazy A N
Published: January 2020

Background

Intestinal homeostasis depends on the interplay between the gut microbiota, epithelium and immune cells. A novel role of Oncostatin M (OSM), a pro-inflammatory cytokine has recently been identified in mouse and human intestinal inflammation. Previous studies have shown OSM as a key driver of chronic inflammation in anti-TNF-α-refractory colitis. A single-nucleotide polymorphism in the human OSM genetic locus is strongly associated with risk of developing inflammatory bowel disease (IBD), thus, biological therapies targeting OSM could have therapeutic potential. Our project aims to explore the impact of OSM on intestinal barrier function in health and disease.

Methods

To evaluate the role of OSM in intestinal inflammation, we utilized a combination of in vitro and in vivo techniques. This included the generation of 3D intestinal organoids from mice and patients. Organoids were stimulated with a repertoire of different cytokines to determine the responsiveness of OSM receptor (OSMR) to different cytokine signals using a quantitative-PCR-based approach. For in vivo modelling of disease, the Helicobacter hepaticus colitis model was used, as it combines both immune and dysbiosis-driven aspects of disease. This allowed us to measure OSM and OSMR expression in response to inflammation and within specific organs and cell subsets. Furthermore, RNAscope in situ hybridisation was used to determine the localisation of OSM- and OSMR-expressing cells in inflamed mucosal tissue from colitic mice and IBD patients.

Results

RNAscope in situ hybridisation as well as gene expression analysis have shown that the OSM and OSMR were highly expressed in C57BL/6 mice upon induction of colitis in the H. hepaticus model of disease and in mucosal tissues of IBD patients. In addition, a plethora of pro-inflammatory cytokines were upregulated during colitis, with colitic mice showing increased tissue pathology. Furthermore, FACS analysis shows excessive immune cell infiltration in the spleen, colon and mesenteric lymph nodes of colitic mice.

Conclusion

Our preliminary results have shown that different gut-resident hematopoietic and non-hematopoietic cell types express OSM and OSMR and this expression was modulated by pro-inflammatory cytokines. We therefore hypothesis that OSM might drive distinct transcriptional responses in various gut-resident cell populations. Thus, differential targeting of the OSM receptor might be a potential therapeutic approach in IBD.