bluuebunny/crossref_metadata_2025_split huggingface dataset:
P029 Histopathological characterization of a chronic DSS-induced mouse model of IBD with intestinal fibrosis
Authors: Nielsen M H , Zachariassen L F , Sárvári A K , Marstrand-Jørgensen A B , Hansen H H , Feigh M 
Published: January 2023

Abstract

Background
Inflammatory bowel disease (IBD) comprises a group of intestinal disorders, including ulcerative colitis and Crohn's disease. Intestinal fibrosis, as result of chronic inflammation, is a common complication in IBD. High treatment failure rates associated with existing interventions indicate a high unmet need for more effective drugs to improve the management and outcomes of IBD. Consequently, translational animal models of IBD demonstrating chronic, progressive colonic fibrosis are important tools in preclinical drug discovery for IBD. The aim of the present study was to characterize intestinal histopathology in a chronic DSS-induced mouse model of IBD.

Methods
Ten-weeks-old male C57BL/6JRj mice were randomized into study groups based on body weight and received 3 cycles of DSS (2.5 % w/v) in the drinking water (DSS-IBD) or normal water (CTRL). Terminal endpoints included colon morphometry and quantitative histological markers of inflammation and fibrosis assessed in proximal, middle and distal colonic segments. Using RNA sequencing, transcriptome signatures were profiled in the middle colon segment.

Results
Compared to healthy controls, the chronic DSS-IBD mouse model demonstrated hallmarks of IBD, including mild-to-moderate weight loss and colonic hypertrophy. Histopathological analysis indicated increased deposition of inflammatory (CD45 and CD11b), fibrogenesis (alpha-smooth muscle actin) and fibrosis markers (collagen 1a1, Picrosirius red) in the middle and distal colon. Severe fibrosis was observed throughout all layers of the middle colon. Histological findings were supported by significantly upregulated gene expression markers of inflammation and fibrosis.

Conclusion
The DSS-IBD mouse is a translational preclinical model suitable for testing novel drug therapies for IBD with intestinal fibrosis.
P034 A common IL2RA polymorphism alters natural killer cell immunophenotype and function in Crohn’s disease, with implications for IL-2 based therapy
Authors: Clough J , Roberts L , Goldberg R , Lord G , Irving P 
Published: January 2023

Abstract

Background
The single nucleotide polymorphism (SNP) rs61839660 lies within the IL2RA (CD25) intronic enhancer and has been identified as a causal variant in Crohn’s disease (CD), with a heterozygote frequency of 16.8% in a European population. CD25 is the high-affinity receptor for IL-2, and is constitutively expressed on regulatory T cells (Tregs). Low-dose IL-2 (LD-IL2) is under investigation in IBD, under the premise that it will expand a group of tolerising Tregs. However, finding a therapeutic dose which activates Tregs without activating effector T cells (Teffs) has proved challenging and activation of CD56dim NK cells has been linked to adverse events in trial subjects. We have demonstrated that the rs61839660 risk (T) allele carriers exhibit higher CD4+ T cell CD25 expression, enhanced pSTAT5 activation on stimulation with IL-2, and greater Teff release of pro-inflammatory cytokines. We sought to understand the impact of the risk allele on NK cell immunophenotype and function.

Methods
We recruited CD patients homozygous major allele (CC), or heterozygous (CT) at the rs61839660 locus. We extracted peripheral blood mononuclear cells and flow-sorted CD56dim (cytotoxic) and CD56bright (regulatory) NK subsets. Cells were stimulated with IL-2, then stained for intracellular pSTAT5. To assess cytotoxicity, NK cells were rested in media either with or without IL-2, before plating with CFSE-labelled K562 target cells. The proportion of K562 cells positive for dead cell marker was assessed for each condition.

Results
There was no difference in CD phenotype or severity between groups, in terms of age at diagnosis, sex distribution, presence of perianal disease or the proportion requiring surgery. CD25 expression was significantly enhanced on CD56Bright NK cells in CT subjects compared to CC subjects (1b). Enhanced CD25 expression was also seen in CD56Dim NK cells, though this did not reach statistical significance (1a). The concentration of IL-2 required to activate 50% intracellular STAT5 was lower in CT subjects’ CD56Bright NK cells compared to those from CC subjects (21.5IU/ml vs. 132.4IU/ml, p=0.039). The cytotoxicity of CT subjects’ CD56dim NK cells was significantly enhanced following exposure to IL-2, compared to CC subjects (1c).

Conclusion
Our findings suggest that risk (T) allele carriers have enhanced responsiveness to IL-2 signalling in NK cells, mediated by increased expression of CD25. We hypothesise that these subjects would be at greater risk of adverse events on exposure to LD-IL2, through enhanced CD56Dim NK cytotoxicity. Our results suggest that screening of subjects entering trials of IL-2-based therapy for the rs61839660 risk variant should be considered, as the threshold for adverse events in these subjects may be lower.
P039 The disappearance of cell death brakes in inflammatory bowel disease: a step towards molecular healing
Authors: Al-Ani A , Samson A , Rickard J , Christensen B , Murphy J 
Published: January 2023

Abstract

Background
The core mechanistic driver underpinning the relapsing and progressive nature of inflammatory bowel disease (IBD) remains unknown with current therapies targeting immune or inflammatory pathways. Our in-depth analysis of regulated death pathways remarkably reveals that cell death brakes are removed in intestinal IBD tissue, indicating cells remain “primed” to die regardless of endoscopic and histologic healing. Characterisation of cell death is essential to confirm our hypothesis that disinhibition of programmed cell death is a rudimentary molecular defect in IBD. Restoration of cell death brakes may be intrinsic to molecular healing and novel drug development.

Methods
Intestinal biopsies are collected from inflamed, marginal and non-inflamed regions during endoscopy for patients with IBD and matched with those without IBD (“controls”). Tissue is processed to quantify cell death signals and changes to the local microbiome using immunoblotting, immunohistochemistry, immunofluorescence, organoid preparation and RNA sequencing (Fig. 1). Laboratory data are correlated with clinical, biochemical, endoscopic and histologic parameters.

Results
To date, 59 patients have been recruited: 29 controls; 31 IBD (13 Crohn’s disease, 16 ulcerative colitis, 2 pouchitis). Interim analyses of 20 patients show loss of cell death brakes (cIAP1, cIAP2, cFLIP) suggesting disinhibition of cell death even in endoscopically and histologically normal IBD tissue (Fig. 2). Concurrently, there is increased apoptosis (characterised by cleaved caspase-3) in IBD intestinal tissue regardless of inflammatory activity (Fig. 3). Necroptosis, a programmed lytic form of cell death, is active in steady state conditions in controls. Additionally, necroptosis appears markedly elevated in stricturing and penetrating Crohn’s disease compared to other subsets of IBD (Fig. 2).

Conclusion
This study demonstrates for the first time that cellular death brakes are removed in intestinal tissue in IBD, despite deep remission with seemingly effective biologic therapy. Disinhibition of programmed cell death is likely intrinsic to the inflammatory sequence underlying disease recurrence and progression. Restoration of cell death brakes may hence be key to achieving long-term, relapse-free remission and should be considered an aspirational goal for both drug development and achievement of molecular healing. Our results challenge current dogma based on murine data that necroptosis occurs in the absence of apoptosis and that necroptosis is universally increased in all forms of IBD. Instead, we propose that a balance of apoptosis and necroptosis is essential in intestinal homeostasis with endotyping of disease based on cell death profile a feasible goal towards precision medicine in IBD.
P044 Ex vivo effects of infliximab on the long non-coding RNAs expression levels in Crohn′s disease
Authors: Baldan-Martin M , Rubín de Célix C , Orejudo M , Ortega L , Fernández-Tomé S , Soleto I , Ramírez C , Arroyo R , Fernández P , Santander C , Moreno-Monteagudo J A , Casanova M J , Casals F , Casabona S , Becerro I , Marigorta U M , Aransay A M , Bernardo D , Chaparro M , Gisbert J P 
Published: January 2023

Abstract

Background
In recent years, evidence shows that long non-coding RNAs (lncRNAs) are key regulators of gene transcription and play important roles in the pathogenesis of inflammatory bowel diseases. LncRNA are involved in regulation of intestinal epithelial cell apoptosis, cell-cell interactions, enhancing inflammation, among others. Biological therapies, which are considered the most potent for disease control, only benefit one-third of patients. For this reason, a deeper understanding regarding the mechanisms by which biological drugs elicit their effect on intestinal mucosal is needed. Hence, we aimed to unravel the ex vivo modulator effect of infliximab on the lncRNAs expression in intestinal biopsies from patients with Crohn′s Disease (CD).

Methods
We performed an unbiased transcriptomic analysis of intestinal biopsies from the ileum and colon from 30 patients [active CD = 10, quiescent CD = 10, healthy controls (HC) = 10] to identify lncRNA differentially expressed in the setting of infliximab modulation (Figure 1). Endoscopic biopsies were cultured with or without infliximab, and the transcriptome was determined by stranded total-RNAseq (reagents from Illumina Inc.). We used different databases (Ensembl Biomart, RNAcentral and ToppGene) to search for non-annotated lncRNA information and data on the location (cellular component), biological process and molecular function of differentially expressed lncRNAs.

Results
Transcriptomic results revealed a widespread dysregulation of lncRNAs in ileum biopsies from patients with active CD, quiescent CD and HC compared to the colon at baseline and after infliximab culture (Figure 2). These differentially expressed lncRNAs were enriched in such pathways as proliferation, apoptosis, migration, inflammatory response of fibroblasts, response to wounding, posttranscriptional regulation of inflammatory genes and activation of the mitogen-activated protein kinase signaling pathways. Regarding the effect of infliximab according to intestinal location, presence of disease and activity, no significant lncRNAs were identified in the different study comparatives.

Conclusion
We have characterized the basal transcriptomic landscape of lncRNAs in patients with CD (active and quiescent) and HC both in ileum and left colon. However, we have not found differential lncRNA expression due to the effect of infliximab, suggesting that the location (ileum or colon) is more relevant when analyzing differences in lncRNA expression in the intestinal tissue.
P049 Proteomic characterization of serum extracellular vesicles from newly diagnosed patients with inflammatory bowel disease
Authors: Soleto I , Baldan M , Ramírez C , Orejudo M , García S , Mercado J , Azkargorta M , Lloro I , Ortega Moreno L , Aldars Garcia L , Riestra S , Rivero M , Gutiérrez A , Rodríguez-Lago I , Fernández L , Ceballos D , Benítez J M , Aguas M , Bastón Rey I , Bermejo F , Casanova M J , Lorente R , Ber Y , Ginard D , Esteve M , Elortza F , Gisbert J P , Martin-Cofreces N , Chaparro M 
Published: January 2023

Abstract

Background
The etiology and pathogenesis of inflammatory bowel disease (IBD), Crohn’s disease (CD), and ulcerative colitis (UC) are complex and the mechanisms that lead to the development of these diseases remain unclear. Extracellular vesicles (EVs) are small particles covered with a cell membrane, originating from the emitting cell, excreted into the extracellular medium, and subsequently captured by receptor cells. EVs have a role in multiple diseases and they could also have a function in IBD pathogenesis. Therefore, the analysis of EVs isolated from the serum of newly diagnosed IBD patients (before starting any treatment) may represent an appropriate experimental approach to elucidate their role in IBD pathogenesis. We aimed to evaluate EVs’ composition and potential effects in the pathogenesis of IBD.

Methods
The protein content of EVs isolated by size exclusion chromatography from the 500 µl of serum from 100 patients with IBD (50 patients with CD and 50 patients with UC) recently diagnosed and 50 healthy controls (HC) was characterized by proteomics and their size and concentration in serum by Nanoparticle tracking analysis (NTA). The biological function of EVs and alterations in signaling pathways related to IBD were determined using Ingenuity Pathways Analysis (IPA) to analyze the OMICs results.

Results
A total of 1,100 proteins have been identified, of which 105 proteins were differentially expressed by patients with CD versus HC, 111 proteins by patients with UC versus HC, and 32 proteins by patients with UC versus CD. IPA analysis revealed that proteins carried in EVs are involved in the dysregulation of immune pathways such as the acute phase response (Figure 1), LXR/RXR activation pathway (Figure 2), and the complement pathway. These pathways were regulated differentially in IBD patients compared with HC, but also when the CU group was compared with the CD group (Figure 3) being upregulated nuclear receptors signaling and cytotoxicity pathways.

Conclusion
EVs carry proteins that can be involved in the dysregulation of the immune system in IBD; this effect would be different in UC and CD since their EVs show a differential profile. Consequently, EVs may play role in IBD pathogenesis and source of strong biomarkers candidates for diagnosis in UC and CD. Further studies on their specific function during IBD are warranted.
P051 Immune phenotypes in acute severe ulcerative colitis
Authors: Meringer H , Canales-Herrerias P , Jha D , Livanos A , Jougon J , Cossarini F , Tankelevich M , Kayal M , Colombel J F , Mehandru S 
Published: January 2023

Abstract

Background
Acute Severe Ulcerative Colitis (ASUC) may occur in up to 25% of patients with UC during their disease course. Despite intensive in-hospital management, some patients still require colectomy. We aimed to define the mucosal immune phenotypes related to ASUC that associate with treatment-outcomes.

Methods
Adult patients with ASUC (defined by Truelove and Witts criteria), (n=32) were recruited. UC patients (mild-moderate disease) (n=31) and normal volunteers (NV) (n=27) served as controls. Gastrointestinal (GI) biopsies from the left colon were analyzed by multiparameter flow cytometry (FC) for myeloid cells (classical and non-classical monocytes, dendritic cells), lymphoid cells (T cells, B cells) and innate (Natural Killer) cells. Therapeutic non-response was defined as surgery within 3 months of admission.

Results
Six of the ASUC patients (18.8%) required colectomy during index admission and 8 (25%) within 3 months of admission, i.e. non-responders. Only 14/32(44%) patients were bio naïve on admission. Clinical parameters significantly associated with treatment non-response included lower hemoglobin and albumin levels (Fig. 1A). Significantly higher frequency of classical (CD14+) and non-classical (CD16+) monocytes as well as proliferating CD4+ and CD8+ cells were seen in ASUC compared to NV and UC patients with Mayo 1, 2, 3 inflammation (Fig. 1B). Furthermore, ASUC patients who were non-responsive to medical treatment had higher frequencies of CD14+ monocytes and proliferating (Ki67+) CD4+ T cells in the colon compared to medical treatment responders (Fig 1C).

Conclusion
High frequencies of CD14+ monocytes and proliferating CD4+ T cells in the colon represent distinct immune signatures that relate to ASUC outcomes. Clinical trials targeting adverse clinical and immune phenotype-associated ASUC patients with combination therapies are warranted.
P052 IL-12p70 as a potential driver of pathogenic IL-17+IFNγ+ mucosal memory TIGITneg T cells in a subgroup of severe CD patients
Authors: Heredia M , Barendregt D M H , van den Bogaerdt S , Samsom J N 
Published: January 2023

Abstract

Background
Crohn’s disease (CD) is a chronic disease driven by inflammatory memory CD4+ T cells. Deciphering heterogeneity in underlying immune disease is required for predicting the individual patients’ disease course. We have shown that analysis of circulating CD38+ memory CD4+ T cells, which are enriched for gut-homing, identifies therapy-naïve pediatric CD patients with a more severe disease course. At diagnosis, these patients have increased frequencies of circulating CD38+ memory T cells that do not express TIGIT (TIGITneg). TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains), is a coinhibitory receptor selectively induced on CD4+ T cells with a memory phenotype. In healthy controls, TIGIT+CD38+ memory T cells have an inhibitory function while TIGITnegCD38+ memory T cells are enriched in inflammatory cells. In our cohort of CD patients, circulating inflammatory TIGITnegCD38+ memory T cells had increased frequencies of pathogenic IFNγ +IL-17+-double-producing or Th1* cells. We hypothesize that in these severe CD patients, CD-related inflammatory cytokines selectively increase frequencies of Th1* TIGITnegCD38+ memory T cells.

Methods
We cultured peripheral blood memory CD4+ T cells from healthy individuals in vitro with TCR- and co-stimulation together with different cocktails of CD-related inflammatory cytokines during 72h.

Results
Of all cytokines tested, only cocktails containing IL-12p70 increased TIGITneg cell frequencies within gut-homing CD38+ memory T cells, but not CD38neg memory T cells that are enriched in skin-homing. As hypothesized, IL-12p70-stimulated cultures contained higher frequencies of Th1* cells, expressing both hallmark transcription factors T-bet and RORgt, in TIGITnegCD38+ T cells but not within TIGIT+CD38+ T cells. This selective IL-12p70 responsiveness in TIGITneg cells agreed with the higher expression of the IL-12 receptor b2 subunit in TIGITnegCD38+ compared to TIGIT+CD38+ memory T cells. These data argue that IL12p70 initiates reprogramming of the TIGITnegCD38+ memory T cells to the Th1* phenotype. To assess whether IL-12p70 causes expansion of TIGITnegCD38+ cells or downregulates TIGIT expression on TIGIT+CD38+ cells, we traced fluorescently-labelled TIGIT+ cells during culture. TIGIT+ cells did not downregulate TIGIT, indicating that IL-12p70 increased proliferation of TIGITnegCD38+ cells.

Conclusion
The finding that IL-12p70 drives expansion of TIGITnegCD38+ memory T cells with a pathogenic Th1* phenotype argues that this cytokine may enforce chronic inflammation in these severe CD patients. These analyses pave the way toward deciphering key actors involved in CD inflammation at an individual patient level, needed to tailor novel IL-12/IL-23 targeting therapeutic strategies.
P057 Development and evaluation of the i-Tracker Certolizumab and i-Tracker Anti-Certolizumab kits: rapid and innovative tests intended for monitoring patients treated with Certolizumab.
Authors: Khater G , Noguier G , Daviere S , Guilbert V , Montaillier C 
Published: January 2023

Abstract

Background
Certolizumab is a human monoclonal antibody directed against TNFα. This drug is commonly used for the treatment of patients with inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis or even psoriatic arthritis. Currently, monitoring of Certolizumab plasma levels is available and provides clinicians with relevant information to improve treatment efficacy. Theradiag has just developed new rapid (results in less than 40 minutes) and innovative assays allowing the quantification of Certolizumab: these tests are available on the new innovative i-Track10 chemiluminescence platform and allow the rapid quantification of Certolizumab as well as anti-antibody antibodies. -Certolizumab induced by the patient.

Methods
Analytical performance was studied using two types of samples: human serum samples spiked with Certolizumab or anti-Certolizumab antibodies, and human serum samples from patients with inflammatory diseases treated with Certolizumab (n=56 ). For drug assay, Certolizumab was captured using TNFα-coupled magnetic microspheres and detected using an acridinium ester-conjugated anti-Certolizumab polyclonal antibody. For the assay of anti-Certolizumab antibodies, microspheres coupled to Certolizumab were used. The anti-Certolizumab antibodies thus captured were detected by Certolizumab conjugated to the acridinium ester. Light emission from acridinium ester conjugates is directly related to the amount of Certolizumab or anti-Certolizumab antibodies present in the sample.

Results
The quantification of Certolizumab by the i-Tracker Certolizumab kit shows very good accuracy (the percentages of recovery are between 84% and 102%). The intra-test and inter-test reproducibility is very good (CV &lt;4.8% for the Certolizumab assay and CV &lt;10.6% for the anti-Certolizumab antibody assay). No interference from biological agents (bilirubin, hemoglobin, triglycerides, rheumatoid factors) was detected. The assay measurement ranges are 3 µg/mL to 100 µg/mL for Certolizumab and 10 AU/mL to 1500 AU/mL for anti-Certolizumab antibodies. i-Tracker assays were compared to ELISA-based Lisa-Tracker assays. The correlations are excellent: the Certolizumab assay presents an R² = 0.94 and a slope of 0.92, the anti-Certolizumab antibody assay presents a Spearman coefficient of 0.94.

Conclusion
The i-Tracker Certolizumab and anti-Certolizumab kits are rapid tests (results obtained in less than 40 minutes) and highly accurate. Very good correlations were observed with the Lisa-Tracker tests (ELISA method) already used routinely. i-Tracker kits are effective tools for monitoring patients treated with Certolizumab.