bluuebunny/crossref_metadata_2025 huggingface dataset:
P064 Investigating the genotoxic effect of reduced glutathione, n-acetylcysteine, human umbilical cord-derived mesenchymal stem cell exosomes and let-7 miRNA mimics in human lymphocytes and EpiIntestinal cells
Authors: Adoul Z , Jafarinejed S , Baumgartner A , Isreb M , Liu F , Anderson D , Najafzadeh M
Published: January 2024

Background

The current treatments for IBD frequently do not provide sufficient control over the disease, warranting the investigation of alternative therapeutic options with minimal risk of side effects. In IBD, the overgeneration and insufficient removal of reactive oxygen species (ROS) leads to oxidative stress. Reduced glutathione (GSH) and n-acetylcysteine (NAC) are antioxidants which may possess therapeutic potential in IBD, by modulating endogenous mechanisms that decrease ROS production or increase antioxidant enzymes. Additonally, exosomes from human umbilical cord blood derived mesenchymal stem cells (hucMSCs-exo) may be used to treat IBD. These are a subset of extracellular nanosized membrane vesicles which participate in intercellular communication by delivering their contents, such as functional miRNAs to recipient cells, thereby influencing the physiological and pathological processes in various diseases. The therapeutic potential of exosomes can be enhanced through the overexpression of the let-7 family of miRNAs.

Methods

The EpiIntestinal tissue model is a physiologically relevant predictor of drug-induced GI toxicity. EpiIntestinal cells and lymphocytes from 3 IBD patients were transfected with 50 μL miRNA mimics and inhibitors, and treated with 50μL hucMSC-exo, and 50 μL 1mM GSH and NAC. Next, in comparison to negative controls, single-stranded DNA damage was assessed in peripheral blood mononuclear cells (PBMCs) from IBD patients using the fast microplate DNA damage assay, and double-stranded DNA damage was assessed in EpiIntestinal cells using the alkaline Comet assay. In addition, the viability of healthy and IBD lymphocytes was assessed following treatment with selected concentrations of hucMSC-exo, GSH and NAC.

Results

Double-stranded DNA damage was significantly reduced in EpiIntestinal cells compared to the negative control following treatment with hucMSC-exo, GSH, NAC, and miRNA mimics for let-7a-5p, -7b-5p, -7c-3p, -7d-3p and -7d-5p. In addition, single-stranded DNA damage was reduced in IBD PBMCs compared to the negative control following treatment with hucMSC-exo, and let-7b-5p and -7c-3p mimics, but was increased following treatment with inhibitors for let-7b-5p, -7c-5p and two novel miRNAs. Additionally, GSH, NAC and hucMSC-exo increased cell viability in treated versus untreated lymphocytes from healthy individuals and IBD patients.

Conclusion

Selected concentrations of HucMSC-exo, GSH, NAC and/or let-7 miRNA mimics reduced DNA damage and increased cell viability in treated versus untreated cells which may reduce the risk of colorectal cancer in IBD patients.

Acknowledgements

Our gratitude goes to the MatTek Team for generously granting us a SMI-100-FT EpiIntestinal kit in support of this research.
P083 Dietary-derived ω-3 and ω-6 polyunsaturated fatty acids trigger an inflammatory response in intestinal epithelial cells with chemically inhibited GPX4 activity
Authors: Schwärzler J P , Mayr L , Grabherr F , Meyer M , Jukic A , Tilg H , Adolph T E
Published: January 2024

Background

The incidence of inflammatory bowel diseases (IBD) has increased drastically in recent years, which is mainly explained by environmental factors and changes in our diet. Recent studies indicated that a Western-style diet, characterized by an increased caloric intake and consumption of long-chain fatty acids such as polyunsaturated fatty acids (PUFAs), triggers inflammation in metabolic active tissues, termed metabolic inflammation. We recently demonstrated that ω-3 and ω-6 PUFAs fuel an inflammatory response in intestinal epithelial cells (IECs) with genetically reduced expression of the anti-oxidative enzyme glutathione peroxidase 4 (GPX4), and similarly a Western-style diet enriched with PUFAs triggered severe enteritis in mice with reduced GPX4 specifically in IECs (i.e., Gpx4+/-IEC mice). Here, we investigated if PUFAs similarly trigger an inflammatory response in IECs with chemically inhibited GPX4 activity.

Methods

Experiments were performed with an immortalised murine IEC line (MODE-K) cells. Cells were stimulated with the ω-3 PUFA stearidonic acid (50 µM), the ω-6 PUFA arachidonic acid (20 µM) or a respective control and the GPX4 inhibitor (1S,3R)-RSL3 (RSL3). Cell death, production of inflammatory cytokines, stress at the endoplasmic reticulum (ER) and activation of mitogen-activated protein kinases (MAPKs) were examined.

Results

Treatment with RSL3 induced cell death in IECs in a dose dependent manner. For further experiments, we worked with 20 nM RSL3, as this concentration already induced some amount of cell death but approximately 75% of cells were still viable. Stimulation with both ω-3 and ω-6 PUFAs triggered the production of interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 1 (CXCL1) in RSL3 co-treated IECs. Moreover, co-treating of RSL3 and PUFAs induced ER stress and activation of the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α), an inflammatory signalling hub, in IECs which was paralleled by the activation of MAPKs and especially the c-Jun N-terminal kinase (JNK) branch.

Conclusion

Our findings demonstrate that the chemical inhibition of GPX4 was sufficient to fuel inflammation in IECs in response to PUFAs, which we previously similarly observed in IECs with genetic depletion of Gpx4.
P099 Neutrophil extracellular traps enhance profibrotic activity of intestinal fibroblasts in Crohn's disease through TLR2/NF-kB pathway
Authors: Dragoni G , Ke B J , Picariello L , Ceni E , Mello T , Verstockt B , Abdurahiman S , Biscu F , Innocenti T , De Hertogh G , Martinod K , Galli A , Matteoli G , Vermeire S
Published: January 2024

Background

Neutrophil extracellular traps (NETs) consist of DNA filaments and cytoplasmic protein granules, extruded by neutrophils after PAD4-dependent activation. NETs release occurs early during inflammation in various immune-related diseases, further aggravating tissue injury and contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in Crohn’s disease (CD).

Methods

NETs and activated fibroblasts were labelled by multiplex immunofluorescence staining on unaffected, inflamed and fibrotic ileum derived from patients with stricturing CD undergoing ileocaecal resection. Intestinal fibroblasts isolated from unaffected CD ileum were co-cultured with NETs for 24 hours to analyse their transcriptome and quantify collagen released with SIRCOL®. Fibroblast migratory activity was investigated with a scratch test. Immunofluorescence intensity of collagen and fibroblast activation protein (FAP) was quantified using the Operetta® Analysis System. Transfection of fibroblasts with NF-kB-luciferase reporter plasmid was performed to evaluate the TLR2/NF-kB pathway, and a specific TLR2 inhibitor (TL2-C29) tested. Finally, mice with selective deletion of PAD4 in neutrophils (PAD4fl/flMRP8Cre+) were subjected to chronic Dextran Sulphate Sodium (DSS) colitis to assess the role of PAD4-mediated NETosis in intestinal fibrosis development.

Results

We found spatial tissue distribution of clusters of NETs adjacent to FAP+ fibroblasts in ileal ulcerations in patients with active CD. Transcriptomics demonstrated upregulation of various profibrotic processes in fibroblasts stimulated with NETs, including TGFβ-receptor signalling pathway, positive regulation of collagen metabolic process, and TLR-signalling pathways. This correlated with increased proliferation rate (p<0.0001), slower wound healing capability (p<0.0001) and higher collagen release in the medium (p<0.01) in the NET-treated group, as well as higher expression of collagens and FAP. Among TLR genes, TLR2 was found upregulated after stimulation with NETs (p=0.02). Transfection data showed significant upregulation (p<0.05) of NF-kB in the NETs-treated group, whereas its expression and soluble collagen release decreased with the addition of the TLR2 antagonist TL2-C29. In line, a significantly lower amount of collagen deposition was observed in the colon of PAD4fl/flMRP8Cre+ mice subjected to chronic DSS colitis, as well as modulation of MMP2/TIMP2 balance and reduced fibroblast activation.

Conclusion

NETs may represent an early trigger of intestinal fibroblast activation via the TLR2/NF-kB axis. As NETs are early players during inflammation, blocking PAD4 may reduce inflammation and thus fibrogenesis in CD.
P101 Investigating the role of gut eukaryotic virome in contributing to colorectal cancer carcinogenesis
Authors: Facoetti A , Massimino L , Cagliani S , Spanò S , Nicolò S , Errico C , Parigi T L , Danese S , Ungaro F
Published: January 2024

Background

Eukaryotic-targeting viruses have recently attracted interest in gastrointestinal diseases such as ulcerative colitis (UC) and colorectal cancer (CRC). UC and CRC share several factors influencing their etiogenesis, including intestinal dysbiosis. Our group has recently published a work pinpointing a gut virome-associated Orthohepadnaviridae protein, the Hepatitis B protein X (HBx) to correlate with UC pathogenesis and promote intestinal inflammation in mice by disrupting the epithelial barrier and shaping the gut mucosal immune environment, independently of the microbiota. Moreover, we showed that HBx induces DNA damage-related biological processes and active processes related to the Wnt pathway known to be involved in CRC carcinogenesis. Based on this evidence we hypothesize that HBx may fill the gap existing between intestinal inflammation and CRC onset by stimulating pro-inflammatory and/or pro-carcinogenic effects.

Methods

To verify this hypothesis, we evaluated the presence of HBx-transcript in CRC-derived mucosal samples. HBx positivity of CRC patient-derived mucosa was assessed by RT-PCR. Caco-2 cells, an in vitro model of the gut barrier, were transduced with HBx protein-encoding lentiviral vectors or GFP as control and were characterized for their carcinogenic and pro-inflammatory phenotypes by FACS analysis. C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx- or the control. FACS analysis and transcriptomic analysis were performed on mice colonic samples.

Results

Metatranscriptomic analysis revealed that HBx was highly enriched in CRC-derived mucosal samples as compared to healthy individuals, strengthening the link between a virome protein-induced inflammation and tumor development in patients. Moreover, HBx overexpression in Caco-2 cells directly provokes DNA damage, as assessed by the significant increase of γH2AX+ cells. Furthermore, since HBx was recently shown by our group as capable of inducing colitis-like symptoms in mice, we investigated whether this viral protein may induce colitis-associated cancer over time. Interestingly, experiments conducted at longer time points (from 40 to 60 days long observation) revealed that mice developed polypoid structures in the colonic mucosa, characterized by a decreased abundance of T cells, including effector T and NKT cells. Of note, in vivo HBx modulated the expression of genes involved in the activation of the proto-oncogene PIM3, further supporting the role of this viral protein on CRC onset.

Conclusion

This study introduces unconventional and innovative insights for giving CRC studies a new direction for scientific and clinical investigations, looking at the gut virome as an active component and not as a mere bystander entity of the microbiota.
P111 Dietary emulsifier κ-carrageenan does not directly affect intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease
Authors: Vissers E , Arnauts K , Giorio L , Zadora W , Wellens J , Verstockt B , Ferrante M , Vermeire S , Matthys C , Sabino J
Published: January 2024

Background

The dietary emulsifier carrageenan (CGN) is widely used to optimise the texture of processed foods such as ice cream, plant-based milk and processed meat products. However, CGN is suggested to play a role in IBD development by exerting pro-inflammatory effects and increasing intestinal permeability, as shown in immortalized cell lines and murine models. Specifically kappa-carrageenan (κ-CGN) has been shown to disrupt barrier function of intestinal epithelial cells. In this study, we investigated if κ-CGN can directly increase intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease (CD).

Methods

Organoid cultures were derived from colonic biopsies of 5 patients with CD (Table 1) and seeded on Transwell® inserts to obtain epithelial monolayers. Two independent experiments were performed per patient (n = 10). Once a confluent monolayer was formed, non-inflamed and inflamed conditions were established by adding regular culture medium or inflammatory stimuli (100 ng/mL TNF-α, 20 ng/mL IL-1β, 1 µg/mL flagellin) to the basolateral side, respectively. The next day, both inflamed and non-inflamed monolayers were stimulated with 100 µg/mL κ-CGN on the apical side, or with regular culture medium as control. The basolateral side was also renewed with either the inflammatory stimuli or regular culture medium. Transepithelial electrical resistance (TEER) was measured after 24 and 48 hours of κ-CGN exposure to study permeability of the epithelium. Relative TEER (% change compared to baseline) was compared between controls and κ-CGN stimulated conditions (repeated measures ANOVA, Šídák's multiple comparisons correction).

Results

As expected, exposure to inflammatory stimuli on the basolateral side resulted in a significant reduction of TEER of the monolayers after 48 hours (+ 24 hours pre-stimulation), compared to the non-inflamed conditions (-22.54 ± 4.557 %, p<0.0001, paired t-test) (Figure 1A). After 24 hours of stimulation with κ-CGN, TEER did not significantly change in both the non-inflamed (p=0.90) and inflamed conditions (p=0.80), compared to the control conditions. Also after 48 hours, exposure to κ-CGN did not significantly affect the TEER in both non-inflamed (p=0.94) and inflamed (p=0.74) conditions (Figure 1B). RNA analysis of the organoid-derived monolayers is ongoing to explore the inflammatory potential of κ-CGN and possible effects on mucus production.

Conclusion

The dietary emulsifier κ-CGN does not directly alter the permeability of the intestinal epithelium of CD patients. Further analysis of gene expression of inflammatory markers and mucus production is ongoing. Possible indirect effects, through alterations of the microbiota or immune cells, should still be explored.
P131 Therapeutic effects of KPG-818 in a trinitrobenzene sulfonic acid - induced mouse Crohn's/Ulcerative Colitis model
Authors: Liao B , Hao K
Published: January 2024

Background

Immunomodulatory drugs have the potential therapeutic benefit not only in malignancy but also in diseases characterized by an inflammatory phenotype. In inflammatory bowel diseases (IBD), colonic inflammation results from dysregulation of the mucosal immune responses to intestinal flora together with genetic and environmental factors. Inflammatory cytokines are implicated in pathogenesis of IBD. Treatments for IBD that improve clinical outcomes and limit adverse effects are needed.

KPG-818 is a novel generation of lenalidomide derivative and small molecule immunomodulatory drug. KPG-818 is currently under clinical development for therapy of multiple myeloma, lymphoma (phase 1), and systemic lupus erythematosus (SLE) (phase 2a). Remarkable immunomodulatory effects of KPG-818 are observed in SLE patients. In preclinical studies, KPG-818 significantly inhibits inflammatory cytokines interleukin-6 and TNF-α in human peripheral blood mononuclear cells, inhibits tumor growth in multiple myeloma and lymphoma xenograft tumor models. Here we report the significant therapeutic effects of KPG-818 in a chronic Crohn’s/ulcerative colitis mouse model.

Methods

Trinitrobenzene Sulfonic Acid (TNBS) was intra-colonially administered to induce Balb/c mouse Crohn’s/ulcerative colitis model at 50 mg/kg, once a week for 4 weeks. KPG-818 was orally administered at doses of 0.03, 0.12, 0.4, and 1.0 mg/kg, once a day for 6 consecutive weeks. Upadacitinib served as a positive control drug. Vehicle solution served as a negative control. To evaluate the therapeutic effects of the KPG-818, disease activity index (DAI) of ulcerative colitis, the degree of weight loss, fecal morphology, and rectal bleeding were scored once every other day for six consecutive weeks. At the end of the study, colonic samples were collected for gross observation and scoring of macroscopic damage, measurement of length and weight, and histopathological diagnosis and scoring. At the end of study, peripheral blood samples were also collected for analyses of immune cells and cytokines.

Results

Compared to the vehicle control, KPG-818 significantly improved the body weight loss, disease activity of ulcerative colitis, gross degree of colorectal injury, pathological lesions of colorectal tissue, inhibited the inflammatory cytokines in a dose-response manner. The effective dose was as low as 0.03 mg/kg. KPG-818 showed similar therapeutic effects to Upadacitinib.

Conclusion

In this mouse model of chronic Crohn’s/ulcerative colitis, KPG-818 showed remarkable therapeutic effects in a dose-dependent manner. These data encourage the clinical evaluation of KPG-818 in inflammatory bowel diseases.
P151 Characterization of platelet activation profile in Inflammatory Bowel Disease
Authors: Rueda Garcia J L , Martín Arranz E , Ramírez García L , Suárez Ferrer C , Sánchez Azofra M , Poza Cordón J , Amiama Roig C , Noci Belda J , Perosanz L , Cubillo García C , García-Rojas C , Silva K , Monzón Manzano E , Arias-Salgado E G , Acuña P , Butta N , Martín Arranz M D
Published: January 2024

Background

Platelet-related markers associated with a procoagulant state have been described for Immune-mediated Inflammatory Diseases (IMIDs), but a broader profiling of these abnormalities in Inflammatory Bowel Disease (IBD) is lacking.

Methods

Cross-sectional study to describe and compare platelet biomarkers of IBD outpatients, both Crohn’s Disease (CD) and Ulcerative Colitis (UC), vs healthy controls (HC).

Patients over 18 years old with a diagnosis of CD or UC according to the European Crohn’s and Colitis Organization (ECCO) criteria were included. Fecal calproctectin (Fcal) >=150ug/g was used as a cutoff level for active disease. Exclusion criteria for IBD patients are listed under Table 1.

Fibrinogen (FBG) receptor on platelet’s surface was determined with anti-CD41 and anti-CD61 monoclonal antibodies (mAbs); and von Willebrand factor receptor with anti-CD42a and anti-CD42b mAbs.

Platelet activation markers [TRAP and ADP-induced activation of fibrinogen (FBG) receptor, and P-selectin (Psel) and CD63 exposure] were determined, respectively, through binding of PAC1, anti-P-selectin and anti-CD63 (mAbs).

All variables were determined by flow cytometry.

Results

55 patients (27 CD, 28 UC) and 75 HC have been included. 43 IBD patients had active disease (21 CD, 22 UC). Complete baseline characteristics of the IBD population are described in Table 1.

Basal Psel (% of positive cells) was increased in CD vs UC (median [IQR]: 5.64 [4,4-10,4] vs 2,9 [1,8-6,8], p=0,02) and vs HC (3,5 [1,4-6,8], p=0,008). ADP-induced Psel was increased in CD vs HC (mean, SD: 49.92, SD 12.75 vs 41.943, SD13.15, p=0,007). Basal CD63 (% of positive cells) was significantly higher in active IBD vs HC (W = 1143, p = 0.02) and inactive IBD vs HC (W = 528, p= 0.006). CD42a (% of positive cells) was decreased in IBD vs HC (W = 1128.00, p=0.007) and CD vs HC (W = 777.00, p =0.04). CD42b (% of positive cells) was also decreased in IBD vs HC (mean, SD: 106.9 SD, 17.32 vs 115.7, SD 28.42, p=0,01) and CD vs HC (104.06 SD 14.49, p=0,03). No differences in basal or TRAP/ADP induced PAC binding, and in CD41 or CD61 expression were observed among groups. Multiple linear regression showed association of Fcal with basal Psel in IBD patients (p=0,02); and Spearman’s correlation showed negative moderate correlation between Fcal and basal Psel (r=0,3; p=0,003)

Conclusion

Features of platelets in IBD suggest increased platelet degranulation and reduced vWF receptor with regards to healthy controls, specially in CD, but no differences in FBG-related receptors. Inverse moderate correlation between Fcal and Psel in IBD patients suggests depletion of intra-platelet granules at higher levels of gut inflammation.
P154 Histological disease progression and ALK5i therapeutic efficacy in a chronic DSS-induced mouse model of IBD with intestinal fibrosis
Authors: Nielsen M H , Zachariassen L , Sárvári A K , Madsen M R , Hansen H B , Fink L N , Fuchs B C , Papazyan R , Aymeric R , Feigh M
Published: January 2024

Background

Inflammatory bowel disease (IBD) comprises a group of intestinal disorders, including ulcerative colitis and Crohn's disease. Intestinal fibrosis, as a result of chronic inflammation, is a common complication in IBD. Many patients do not respond or do not have a sustained response to existing treatments, highlighting the large unmet need for more effective drugs in the management of IBD. Translational animal models demonstrating chronic, progressive colonic fibrosis are important tools in preclinical drug discovery for IBD. The aim of the present study was to characterize histological disease progression and therapeutic efficacy of a TGF-β type I receptor inhibitor (ALK5i) in a novel chronic DSS-induced mouse model of IBD.

Methods

10 week old male C57BL/6JRj mice received 3 cycles of DSS (2% w/v) administration in the drinking water (DSS-IBD) or normal water (CTRL). Study groups were terminated at week 1, 3 or 6. Groups terminated at week 6 received twice daily oral dosing with vehicle or ALK5i (30 mg/kg, BID). Terminal endpoints included distal colon morphometry, quantitative histological markers of inflammation and fibrosis as well as colon transcriptomics.

Results

Compared to healthy controls, the chronic DSS-IBD mouse model demonstrated clinical and histological hallmarks of progressive IBD, including mild-to-moderate weight loss, colonic hypertrophy with sustained inflammation and fibrosis in the mucosa and submucosa areas. ALK5i treatment increased colon weight/length ratio while significantly reducing fractional (%) area of alpha-1 type I collagen in the colonic mucosa and submucosa. The histopathological phenotype was supported by corresponding regulations in gene expression markers of colonic inflammation including ptprc (cd45), tnf and nos2. Alk5i therapy did not influence gene expression markers of fibrosis, suggesting that improved relative (%) levels of collagen were driven by Alk5i-stimulated tissue volume increases. Further studies are needed to establish if TGF-beta inhibitors have antifibrotic effects in the chronic DSS model of IBD complicated by fibrosis.

Conclusion

The DSS-IBD mouse model is a preclinical model with features of progressive IBD suitable for testing novel anti-fibrotic drug therapies targeted for IBD patients.
P161 The use of Novel Natural Treatments for Inflammatory Bowel Diseases
Authors: Najafzadeh M , Najafzadeh Mojgan , Adoul Zahra , Jafarinejad Shohreh , Baumgartner Adi , Reynolds Domenic , Isreb Mohammad , Ghaderi Rojan , Sefat Farshid , Heidari Saeed , Anderson Diana
Published: January 2024

Background

Background: Free radicals and reduced antioxidant levels lead to oxidative stress, which plays a role in causing Inflammatory Bowel Disease (IBD). IBD is a chronic gastrointestinal autoimmune condition that involves an abnormal immune response. This includes Crohn's disease (CD) and ulcerative colitis (UC). In the previous studies, we demonstrated the antioxidant effect of Inonotus obliquus (IO) and flavonoids such as quercetin and epicatechin on lymphocytes from IBD patients. In vitro studies have shown that Chaga extract is effective in reducing oxidative stress in lymphocytes of both healthy individuals and those with IBD. This suggests that Chaga extract may be a useful supplement for anyone looking to inhibit oxidative stress.

Methods

Methods: In our current study, Cord Blood Stem Cells -derived Exosomes (CBSC Exo) and their synthesised miRNAs were investigated as antioxidant/anti-inflammatory elements on lymphocytes and 3D intestinal epithelial model. There were seven miRNAs found in CBSCs Exo, and among them were two miRNAs that were identified as novel. The Peripheral Blood Monocyte Cells (PBMCs) from IBD patients and healthy individuals after challenging with H2O2 or Interleukin 6 were treated with CBSCs Exo, transfected with miRNA novels or both. The treatment method also was applied to the EpiltestinalTK 3D model from Mattek. Various techniques were utilised, including the Comet assay, Fast Microplate DNA damage assay, and CCK8.

Results

The findings of the study indicate that the administration of CBSC-derived Exo, Let-7c-mimic miRNA, Let7bSb miRNA, as well as Novel 1 and Novel 2 miRNAs on the 3D model intestine, significantly decreased the level of DNA damage when compared to the positive control H2O2 and IL6, as well as the untreated samples.

Conclusion

Conclusions: In conclusion, certain natural substances like flavonoids, Inonotus obliquus, and exosomes derived from CBSCs may have an advantage in decreasing significantly oxidative stress and DNA damage in IBD Peripheral Blood Mononuclear Cells (PBMCs) and inflamed intestinal 3D models.
P167 Remodelling of the colonic molecular landscape in patients failing anti-TNF therapy highlights dysregulation of the extracellular matrix in ulcerative colitis
Authors: Cozzetto D , Thomas J , Saifuddin A , Powell N
Published: January 2024

Background

In patients with ulcerative colitis (UC) who previously failed anti-TNF therapy, subsequent treatment with other advanced therapies is generally less effective than they are in anti-TNF naïve patients. Since anti-TNF drugs are frequently used first-line in UC, drug sequencing depreciation poses a major clinical challenge. To understand potential underlying mechanisms, we compared the clinical and molecular phenotype of UC patients who failed anti-TNF therapy with those who were anti-TNF naïve.

Methods

Clinical information and mucosal gene expression profiles were collected from 262 anti-TNF naïve and 275 anti-TNF exposed individuals with moderate to severe UC enrolled in the UNIFI clinical trial programme. We assessed differences between the groups in terms of clinical parameters, gene expression levels, pathway and transcription factor activity, as well as cellular composition.

Results

Anti-TNF exposed patients had significantly increased disease duration (p=0. 000009), CRP (p=0.03), and histological severity[PN1] (p=0.002). After adjusting for these factors, we identified 1,592 genes differentially expressed (FDR < 0.05), although the expression changes were quite modest. Pathway analysis linked previous anti-TNF exposure to significant downregulation of TNF and NFkB signalling and up-regulation of extracellular matrix components, stroma-related signatures, and growth factor receptor (EGFR, MAPK, PI3K) and TGFb signalling. These data were in line with results from cellular deconvolution methods, which showed an increase in stromal cell abundance and T cell depletion following anti-TNF failure. Gene regulatory network inference suggested that the observed gene expression differences were driven by increased activity of many transcriptional regulators, including those downstream of RUNX1 and RUNX3.

Conclusion

In patients with active UC, prior anti-TNF failure is associated with molecular remodelling of the colon characterised by dysregulated ECM organisation and shifts in stromal compartments. These data provide new insights into potential mechanisms of drug sequencing depreciation and could inform novel strategies to overcome this challenging clinical phenomenon.
P170 SLC7A5 promotes the progression of Ulcerative Colitis by regulating amino acid transport and intestinal epithelial autophagy
Authors: Shi Y , Wang F , Hou L , Zhou H , Wu T , Li R , Li X , Liu X , Fan Z , Zhang T , Da Y , Liang J
Published: January 2024

Background

IBD has become a common chronic intestinal disease in China. Considering the important role of amino acid metabolism in inflammatory diseases, inhibition amino acid metabolism of inflammatory sites may be effective in alleviating UC (ulcerative colitis). SLC7A5 is involved in the progression of UC, but the mechanism of its functional regulation still needs to be investigated. In this study, we aimed to clarify the link among SLC7A5 expression in UC, mTOR pathway activation and immunoregulation.

Methods

We previously discovered through high-throughput screening that SLC7A5, as a cellular amino acid transport carrier, is significantly elevated in the colonic tissues of UC patients; in vivo experiments have demonstrated for the first time that JPH203, a specific inhibitor of SLC7A5, alleviates DSS-induced intestinal inflammation and inhibits mTOR pathway activation to promote intestinal autophagy. This study utilized model animals, transcriptomics, amino acid-targeted metabolomics assays, and macro-genome sequencing to elucidate the mechanism of SLC7A5-regulated amino acids in mTOR pathway activation and intestinal autophagy dysfunction.

Results

SLC7A5 expression is increased in the colon of UC patients and DSS-induced mice lesions. SLC7A5 inhibitor (JPH203) restrained the inflammatory responses induced by DSS. SLC7A5 deletion or inhibition dampens the release of IL-1β, IL-18, and IL-23 and the production of ROS in the LPS-induced FHC and RAW264.7 cells. Moreover, upregulating SLC7A5 expression induces mTOR signaling pathway activation in FHC cells. Deletion or inhibition of SLC7A5 efficiently blocks the SLC7A5-dependent amino acid transport, inhibits the mTOR activation, and results in the activation of autophagy.

Conclusion

Targeting SLC7A5-mediated amino acid uptake is a potentially useful immunosuppressive strategy to regulate colonic inflammation through mTOR pathway and autophagy. This study is expected to reveal the intrinsic factors of metabolic disorders promoting the UC progression from the perspective of amino acid metabolism, and to lay a new theoretical and experimental foundation for potential UC treatment.
P171 Rescuing Intestinal Stem Cells via TMEM219 inhibition promotes mucosal healing in Crohn’s disease
Authors: D'Addio F , Canducci F , Marin V , Assi E , Maestroni A , Nardini M , Petrazzuolo A , Loretelli C , Usuelli V , Moufida B N , Sampietro G , Ardizzone S , Fiorina P
Published: January 2024

Background

Intestinal mucosa regeneration is disrupted in inflammatory bowel disease as Chron’s disease, but whether this relies on a defect in intestinal stem cells remains to be established.

Methods

Transcriptome and receptome profile in Crohn’s disease bioptic samples demonstrated a defect in intestinal stem cells (ISCs). The TMEM219 knockout mouse, in which TMEM219 expression is abrogated on ISCs was tested in dextran sulfate sodium (DSS) acute colitis model. DSS-induced chronic colitis was also used to confirm in vivo relevance for TMEM219 blockade in mucosal recovery.

Results

An increased intestinal stem cells death and dysfunction in colonic samples obtained from patients with Crohn’s disease was observed, which is controlled through the death factor TMEM219. Based on a receptome analysis, we documented large alterations in the expression of the death receptor TMEM219 in patients with Crohn’s disease, particularly in those with refractory disease and/or non responders to conventional therapy, which were paralleled by altered peripheral levels of the TMEM219 ligand, insulin-like growth factor binding protein 3 (IGFBP3). Pharmacological blockade of the IGFBP3/TMEM219 axis restored the self-renewal abilities of mini-guts generated from patients with Crohn’s disease in vitro, ameliorated DSS-induced colitis in vivo and favored mucosal healing. Genetic deletion of TMEM219 in intestinal stem cells in newly generated TMEM219flflLGR5cre mice restored mucosal regenerative abilities both in vitro and in vivo.

Conclusion

Our findings demonstrate that genetic and pharmacological TMEM219 blockade re-establishes intestinal self-renewal properties and offers a therapeutic opportunity for mucosal healing in inflammatory bowel disease.
P180 Novel biomarkers associated with inflammatory bowel disease
Authors: Baldan-Martin M , Lloro I , Azkargorta M , Orejudo M , Ramírez C , Soleto I , Mercado J , Suárez-Trujillo F , Sánchez-Sánchez C , Gordillo-Vélez C H , Riestra S , Rivero M , Gutiérrez A , Rodríguez-Lago I , Fernández-Salazar L , Ceballos D , Benítez J M , Aguas M , Bastón Rey I , Bermejo F , Casanova M J , Lorente R , Ber Y , Royo V , Esteve M , Elortza F , Gisbert J P , Chaparro M
Published: January 2024

Background

Inflammatory bowel disease (IBD) is a chronic multi-factorial disease characterized by inflammation of the gastrointestinal tract. Currently, the mechanisms underlying the disease are far from being elucidated. Thus, the study of proteins involved in its pathogenesis would allow further insights into the molecular mechanisms underlying IBD.

Methods

The serum and intestinal mucosa (ileum and left colon) proteomic profiles of patients with Crohn´s disease (CD) and ulcerative colitis (UC) and healthy controls (HC) were analyzed using a label-free quantitative proteomics approach (Table 1). Data mining and pattern recognition techniques were performed to identify hidden relationships that are not detectable using classical linear classifiers, and thus identify potential markers able to discriminate between the different study groups. Six candidate biomarkers were selected for further validation using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in serum and intestinal tissue samples, respectively (Table 2).

Results

Comparison of serum proteins between UC patients and HC revealed statistically lower levels of protein 1 and 2 and higher levels of protein 3. The comparison of potential biomarkers between CD and HC group showed lower levels of protein 1 and higher levels of protein 3. In addition, protein 4 was differentially upregulated in CD patients compared to UC (Figure 1A). Regarding the intestinal biopsies, protein 5 showed higher intensity in the IHC staining of ileum from CD patients compared to HC, whereas protein 6 was significantly decreased in left colon and ileum from CD and UC patients compared to HC (Figure 1B). These proteins are involved in thyroxine transport, regulation of chemotactic chemokine activity, inflammatory processes, triglyceride homeostasis, oxidative stress and regulation of NF-kappa-B activation in the cytosol.

Conclusion

In this study, we have identified a panel of six potential biomarkers that could help to elucidate mechanisms involved in IBD pathogenesis. Their roles in the pathophysiology need to be clarified in further experiments.
P189 Lifestyle and psychosocial factors in IBD; prevalence and patients’ perspective
Authors: Demers K , Hendrix E M B , Rezazadeh Ardabili A , Bredero Q M , van Bodegraven A A , van der Horst D , Jonkers D M A E , Kimman M L , Mujagic Z , Romberg-Camps M J , Römkens T E H , Scherpenzeel M P , Schroevers M J , Stassen L P S , West R L , Dijkstra G , Pierik M J
Published: January 2024

Background

Lifestyle and psychosocial factors impact mucosal inflammation and subjective well-being in Inflammatory Bowel Disease (IBD) patients. Better understanding of the relevancy of these factors within the IBD population, including prevalence and patients' perspective, may enhance systematic implementation of lifestyle interventions in regular care. This collaborative study with the Dutch patient organisation aimed to estimate the prevalence and patients’ perspectives on lifestyle and psychosocial factors in an outpatient cohort of IBD patients.

Methods

A multicentre cross-sectional study was conducted, enrolling IBD patients who utilized the remote monitoring platform myIBDcoach in 2022. Patients reported on clinical disease activity, lifestyle and psychosocial factors on this platform. A nationwide online survey was distributed by the Dutch patient organisation for IBD in April 2022 to assess patients’ perspectives regarding the impact of lifestyle and psychosocial factors on intestinal complaints, motivation to make changes, and need for and satisfaction with support in this regard from the hospital.

Results

In the multicentre myIBDcoach cohort (n=460), 16.3% adhered to a specific diet (Table 1). Two third (67.4%) did not comply with the Dutch healthy exercise norm and smoking was prevalent in 9.3%. About one-third experienced at least regularly poor sleep (33.8%), at least occasionally emotional distress (33.9%), and high perceived stress (36.7%). In the nationwide survey (n=1148), most (58.3-75.7%) patients believed that stress, unhealthy food, poor sleep, minimal or no exercise, and symptoms of anxiety and depression could lead to intestinal complaints (Fig. 1A). Around 70% of patients were taking action or were motivated to do so regarding their diet, stress, and physical activity (Fig. 1B). About 30% had no intention of addressing social support, and 22.8% were unwilling to change their alcohol use. Less than one-fifth received support from the hospital regarding these various factors (Fig. 1C). When support was received, most patients expressed satisfaction. Although desired, approximately 20% lacked support concerning stress, physical activity, diet, and sleep.

Conclusion

There is considerable scope for improvement in lifestyle and psychosocial factors among individuals with IBD, highlighting the need for systematic integration of these factors into clinical care. From the patient's viewpoint, widespread recognition of the impact of these factors emerged, with most expressing openness to initiating changes across various areas. These insights emphasize the potential for implementing targeted interventions, particularly in areas where patients indicated a need for additional support.
P229 Comparison of Different Histological Scoring Systems for Ulcerative Colitis [Nancy Histological Index, Robarts Histopathology Index and IBD-DCA (Inflammatory Bowel Disease-Distribution, Chronicity, Activity Score)] Among Specialists in GI Pathology and General Pathologists Pre- & Post Workshop
Authors: Loong S , Chen X F , Zhang X , Wan W K , Leow W Q , Kieran S , Lim T K H , Wang L M
Published: January 2024

Background

Histopathology is an emerging treatment target and an important parameter in predicting clinical outcomes of patients with ulcerative colitis (UC). Histological scoring indices Nancy Histological Index (NHI) and Robarts Histopathology Index (RHI) have been developed and validated for UC. We aim to compare the inter-observer reliability of NHI, RHI and the recently described Inflammatory Bowel Disease-Distribution, Chronicity, Activity (IBD-DCA) Score among gastrointestinal (GI) pathologists versus general pathologists pre- and post-workshop to determine the scoring system with the best reproducibility.

Methods

Digital whole slide images of 20 H&E-stained biopsies of ulcerative colitis (UC) cases are scored independently by 6 GI pathologists and 44 general pathologists that attended the Singhealth GI Pathology Course 2023 using NHI, RHI and IBD-DCA on the digital Pathpresenter platform. The 44 course participants performed the scoring before the course and again after attending a 3-hour IBD workshop on the assessment of histological remission using these 3 scoring systems. Data tabulation using “Free Online Surveys” platform is collated and intraclass correlation coefficient (ICC) of each component of the scoring indices is calculated. The ICC values of <0.5, 0.5–0.75, 0.75–0.9, and >0.9, indicate ‘poor’, ‘moderate’, ‘good’, and ‘excellent’ agreement, respectively.

Results

There is good inter-observer reliability for IBD-DCA ‘Activity’ component (ICC 0.85) and NHI (ICC 0.83) among GI pathologists while lBD-DCA ‘Distribution’ component (ICC 0.395) has the poorest agreement. Poor to moderate inter-observer reliability for all 3 scoring systems (ICC 0.4 – 0.6) is seen among the 44 participants pre-workshop with an observed post-workshop improvement, achieving best agreement for the NHI, ‘lamina propria neutrophils’ component of RHI and IBD-DCA ‘activity’ component (ICC 0.7).

Conclusion

GI pathologists are generally consistent in assessing disease activity in UC with good inter-observer reliability for NHI (a scoring system with an emphasis on activity grade) and only the ‘activity’ component of the IBD-DCA. Our study confirms NHI as a reliable scoring system for UC with the best reproducibility and favour its application to assess histological response in routine clinical practice to help gastroenterologists better manage patients with UC. We have also demonstrated that improvement in NHI scoring reproducibility can be achieved after training workshop for pathologists less familiar with the application of IBD histological scoring.
P279 Excessive accumulation of neutrophils in the epithelium, but not lamina propria, predicts resistance to ustekinumab in ulcerative colitis
Authors: Saifuddin A , Powell N
Published: January 2024

Background

Many patients experience primary treatment resistance when treated with ustekinumab for ulcerative colitis (UC). Predictive clinical parameters would allow such individuals to be identified pre-treatment so alternative strategies could be initiated. We explored the utility of the different histological features present in baseline samples to predict ustekinumab resistance.

Methods

We assessed the total Geboes score (TG) (0–22, least to most severe), and the epithelial and lamina propria neutrophil subscores (EpiN and LpN, respectively) (both 0–3) in a subset of patients with moderate-to-severely active UC (n = 448) enrolled in the phase 3 UNIFI study that evaluated the efficacy of ustekinumab therapy. We explored the relationships between histological parameters and clinical indices of activity, including faecal calprotectin (FCP), CRP, Mayo score, and treatment outcomes (clinical remission, endoscopic healing and mucosal healing at week 8, and disease clearance, which combines these all) using Spearman rank correlation. Area under the receiver operating characteristic curve (AUC) analyses were performed (presented with 95% confidence interval, CI). Analyses were performed using R 4.2.3 (Vienna, Austria) and GraphPad Prism (v 9.5.1).

Results

TG correlated highly with EpiN (ρ = 0.804, p < 0.0001) but did not correlate with LpN (ρ = 0.082, p = 0.07). Neither EpiN (ρ = 0.081, p = 0.09) nor LpN (ρ = -0.011, P = 0.82) correlated with FCP, though EpiN correlated weakly with log10FCP (ρ = 0.113, p = 0.017). Full Mayo score correlated with EpiN (ρ = 0.23, p < 0.0001) and TG (ρ = 0.23, < 0.0001).

Of the patients with pre-treatment EpiN of 0, 23.1%, 26.9%, 23.1% and 19.2% experienced clinical remission, endoscopic healing, mucosal healing and disease clearance, respectively. Response rates incrementally reduced as EpiN increased, such that 4.2%, 12.5%, 8.3% and 4.2% of patients with EpiN of 3 achieved these outcomes, respectively. Similar trends were seen with tertiles of TG, though not with LpN. EpiN and TG predict mucosal healing with AUC 0.59 (CI: 0.50 - 0.68; p = 0.044) and 0.61 (CI: 0.52 - 0.70; p = 0.016), respectively. The addition of other clinical variables did not improve performance of the models.

Conclusion

There is weak or insignificant correlation between Geboes score parameters and other clinical indices of UC activity. EpiN and TG, but not LpN, stratify patients’ response to ustekinumab, with almost 6-fold difference in clinical remission between high and low EpiN. TG and EpiN predict resistance to ustekinumab, suggesting that the epithelial inflammatory milieu may be key to driving resistance to ustekinumab and possibly other therapies. This supports recent evidence highlighting the role of neutrophils in resistant UC.
P325 Post-COVID general health symptoms in patients with Immune Mediated Inflammatory Diseases
Authors: Van De Pol N , van der Woude C J , Vis M , van Doorn M , Schrauwen S L , Cetinözman-Teunissen F , West R L , Pan Q , de Vries A C
Published: January 2024

Background

Post-COVID entails persistent symptoms following a presumed or confirmed SARS-CoV-2 infection and has a prevalence of 12.5% in the general population in The Netherlands. The aim of this study is to evaluate post-COVID general health symptoms reported by patients with Immune Mediated Inflammatory Diseases (IMIDs), given the unclear prevalence.

Methods

An online questionnaire was distributed to IMID patients at dermatology, rheumatology and gastroenterology departments in an academic and teaching hospital in Rotterdam, the Netherlands. Only patients on systemic therapy were included. General health symptoms were compared between patients with and without prior SARS-CoV-2 infection. Multivariable logistic regression, adjusted for disease-related factors and known post-COVID risk factors, was performed to assess the association between SARS-CoV-2 infection and symptom occurrence. Additionally, the self-reported prevalence of post-COVID symptoms was assessed by asking patients if they linked their symptoms to the previous SARS-CoV-2 infection.

Results

The final study population comprised 1,518 patients, of whom 58% (n=877) reported ≥ 1 SARS-CoV-2 infections (table 1). More patients with a prior SARS-CoV-2 infection reported general health symptoms compared to those without (56% vs 44%; P<0.001) (figure 1). After adjusting for potential risk factors, including age, sex, smoking, diagnosis, SARS-CoV-2 vaccination status, and immunosuppressive therapy, a consistent significant association was found between SARS-CoV-2 infection and symptoms (OR 1.53; 95%CI 1.23-1.91; P<0.001). Sub-analysis showed a significant association between self-reported COVID-19 severity and symptoms, while presumed SARS-CoV-2 variant (based on date of reported infection) and number of infections were not associated. Fatigue was the most common symptom in both patients with and without a previous SARS-CoV-2 infection, with significantly higher prevalence among patients with previous SARS-CoV-2 infection (42% vs 32%; P<0.001). After adjusting for the potential risk factors, a previous SARS-CoV-2 infection remained significantly associated with fatigue (OR 1.31; 95%CI 1.04-1.64; P=0.021). Of the patients with a prior SARS-CoV-2 infection, 14% (122 out of 877) reported post-COVID symptoms, with fatigue as the most common complaint (78%).

Conclusion

The self-reported prevalence of post-COVID general health symptoms among IMID patients on systemic therapy (14%) aligns with that of the general population. The prevalence of symptoms, particularly fatigue, is elevated in patients with a history of a SARS-CoV-2 infection. Our findings emphasize the importance of incorporating SARS-CoV-2 infection history when evaluating fatigue in IMID patients during outpatient visits.