bluuebunny/crossref_metadata_2025_split huggingface dataset:
P058 The identification of three linear epitopes on infliximab all map to complementarity-determining regions
Authors: Tun G , Ramage W , Hufton S E , Lobo A J 
Published: January 2024

Abstract

Background
Infliximab (IFX) is a mainstay of treatment for inflammatory bowel disease (IBD). Its use is limited by the development of anti-drug antibodies (ADA), which can cause loss of response and adverse drug reactions. The antigenic epitopes have not been clearly defined, and large antigenic areas suggested. The aim of this study was to identify clinically relevant distinct epitopes in IBD patients treated with IFX with ADA and different clinical outcomes.

Methods
Sera were pooled from 8 ADA positive IBD patients (ADA &gt;10 AU/mL; IDKmonitor® Infliximab total ADA) treated with CT -P13 (Remsima® Celltrion Inc., Korea) exhibiting: loss of response, response with therapeutic drug levels, response with subtherapeutic drug levels and adverse drug reactions. A novel whole gene fragment library (GFL) was synthesised using pools of overlapping synthetic oligonucleotides covering the IFX heavy chain and light chain (Twist Bioscience, California, United States). This was cloned into a phage display vector to generate a library &gt;106 clones. Three rounds of selection of the GFL were performed using pooled sera. DNA from the selection outputs were analysed by next generation sequencing (NGS) (Illumina, San Diego, United States). Outputs from one round of selection using individual sera were also analysed by NGS.

Results
ADA ranged 17-&gt;600 AU/mL. 23,383 gene sequences analysed from the GFL showed complete coverage of the IFX gene. After three rounds of selection, DNA sequencing showed the enrichment of three peptides – all overlapping the IFX complementary-determining regions (CDR)- Figure 1. The three peptides enriched: heavy chain peptide 1 included heavy chain CDR 1, light chain peptide 2 overlap light chain CDR 1 and light chain peptide 3 overlap light chain CDR 2. NGS assessment of the outputs from round 1-3 using pooled ADA positive sera confirmed enrichment of the three peptides. NGS analysis of the selections on individual sera showed differences in enrichment profile of the three peptides.

Conclusion
Using ADA positive sera from IBD patients treated with IFX with differing clinical outcomes, three linear epitopes were identified from a novel whole IFX-GFL. All epitopes overlapped with CDRs which is consistent with being potential sites for neutralising ADA. NGS has suggested that individual ADA positive sera have differing recognition profiles for the selected epitopes. Identifying distinct epitopes may lead to the identification of a biomarker for loss of response to infliximab.
P061 The macrophage-specific role of PTPN23 in Acute and Chronic Colitis
Authors: Linzmeier L , Matus V , Determann M , Wilmink M , Schwarzfischer M , Sanchez Alvarez R , Pöhlmann D , Scharl M , Spalinger M 
Published: January 2024

Abstract

Background
Single nucleotide polymorphisms within the gene locus encoding PTPN2, are associated with Crohn’s disease and ulcerative colitis. We have previously shown that, deletion of PTPN2 results in PTPN23 upregulation in intestinal epithelial cells (IEC) in vivo. PTPN23 is known to be involved in endosomal sorting and responsible for epidermal growth factor receptor (EGFR) internalization from the cell surface and thus negatively controls EGFR signalling in epithelial cells. Though PTPN23 is highly expressed in IEC and intestinal macrophages, nothing is known about its function in myeloid cells and its role in intestinal inflammation.

Methods
Experimental colitis was induced in PTPN23fl/flLysMCre+/- (KO) and PTPN23fl/flLysMCre-/- (WT) littermates by administration of 2% DSS in drinking water for 7 days (acute colitis) or for 4 cycles of 1.5% DSS for 7 days followed by 10 days recovery, each (chronic colitis). Immune cells were isolated from spleen and colon lamina propria from KO and WT mice and PTPN23 expression was detected by qPCR of RNA isolated after cell sorting with flow cytometry.

Results
The mRNA expression of PTPN23 in untreated WT mice was significantly higher in macrophages isolated from the lamina propria of the colon than in those isolated from spleen (p=0.0279).
PTPN23fl/flLysMCre+/- (KO) mice were protected from DSS-induced acute colitis when compared to their WT littermates. This was apparent by a lack of DSS-induced weight loss (p=0.02); longer colon length (p=0.01), reduced endoscopic colitis scores (p=0.0016) and lower spleen weights (p=0.0063) in KO mice. In HE staining of colon tissue, the loss of crypts was less pronounced in KO mice. On mRNA level a lower expression of the pro-inflammatory cytokines interleukin 6 (IL-6) (p=0.0117) and tumour necrosis factor α (TNFα) (p=0.0024) was observed in KO compared to WT mice, supporting a reduced inflammatory response.
In chronic colitis the same trends was observed, however the effects were less pronounced. WT mice lost more weight during the DSS cycles compared to KO mice, whereas no difference in weight development was detected between the genotypes in water control groups. Higher MEICSs scores were observed in WT compared to KO mice (p&lt;0.0001) and loss of colonic crypts was more pronounced in HE stained colon samples in WT mice than KO mice.

Conclusion
PTPN23 deletion in myeloid cells results in protection from DSS-induced acute and chronic colitis. Additionally, a higher expression in intestinal macrophages compared to macrophages isolated from the spleen suggest a specific role in the intestinal immune system.
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

Abstract

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

Abstract

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

Abstract

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&lt;0.0001), slower wound healing capability (p&lt;0.0001) and higher collagen release in the medium (p&lt;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&lt;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

Abstract

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

Abstract

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&lt;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

Abstract

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.