bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP73 Predicting endoscopic improvement in Ulcerative Colitis using the Ulcerative Colitis Severity Index (UCSI)
Authors: Wong E C L , Dulai P S , Marshall J K , Jairath V , Reinisch W , Narula N
Published: January 2023

Background

Scoring indices used in clinical trials for moderate-severe ulcerative colitis (UC) may lack specificity as a prognostic tool. We developed and internally validated a prognostic scoring index for UC patients on therapy that considers baseline patient-reported outcomes, biomarkers, endoscopy, and histology for achieving one-year endoscopic improvement (EI).

Methods

This post-hoc analysis of the UNIFI clinical trial (ClinicalTrials.gov identifier: NCT02407236) included 644 patients treated with ustekinumab induction therapy. Data were randomly split into 70% training and 30% testing cohorts. Baseline variables were assessed in multivariate analyses and variables with p <0.05 were assigned weights according to their relative prognostic value for predicting one-year EI (Mayo endoscopic score (MES) ≤1). A cut-off was obtained by calculating the maximum Youden index and validated in the testing cohort.

Results

Prior biologic failure, albumin < 40 g/L, CRP > 5mg/L, Mayo stool frequency (SF) subscore, endoscopic erosions/ulcerations based on the UC Endoscopic Index of Severity, and histologic structural/architectural changes demonstrated significant associations with one-year EI and included in the final model. The Ulcerative Colitis Severity Index (UCSI) was generated (Table 1) and the median UCSI score among all participants was 10.4 (IQR 7-14.4, range: 2.2-20), which had acceptable discriminative ability for one-year EI in the training [AUC: 0.78 (95% CI: 0.70-0.86)] and testing cohort [AUC: 0.76 (95% CI: 0.68-0.85)] (Table 2). Compared to the UCSI, the Mayo score demonstrated poor accuracy [AUC: 0.49 (95% CI: 0.40-0.58)] for predicting one-year EI (p=0.0006). The UCSI also predicted one-year endoscopic healing (MES of 0), clinical remission (total Mayo score ≤2 and no subscore >1), partial Mayo score (PMS) remission (PMS < 2), and PRO-2 remission (SF and rectal bleeding subscore of 0) with significantly greater accuracy compared to the Mayo score. Similar findings were observed when the UCSI was compared with the adapted Mayo score. The maximum Youden index corresponded to a cut-off of 10. Participants with a UCSI ≥10 had a lower probability of one-year EI [27/104 (26%) vs. 40/76 (52.6%)]. Performance characteristics are detailed in Tables 3 and 4.

Conclusion

The UCSI is an internally validated prognostic scoring tool that accurately predicts one-year EI at baseline among patients with moderate-to-severe UC on active therapy. The UCSI demonstrated greater accuracy for one-year EI, EH, and clinical remission compared to the total and adapted Mayo score. The UCSI could improve disease management in UC by stratifying patients based on their predicted likelihood of long-term outcomes.
DOP80 Multi-omic sequencing reveals distinctive gene expression and DNA methylation alterations as potential predictors of primary sclerosing cholangitis development in treatment-naïve paediatric ulcerative colitis
Authors: Lawal O , Rodriguez-Sosa A , McDonnell C , Grant L , O'Brien J , Kel A , Dominik A , Stack R , Yochum G , Koltun W , Doherty G , Hussey S
Published: January 2023

Background

Primary sclerosing cholangitis (PSC) is a progressive choleostatic disease and up to 80% of patients also have ulcerative colitis (PSC-UC). This presents a clinical challenge owing to difficulty in diagnosis and increased risk for developing cancer. While several multifactorial processes including inflammation and microbial dysbiosis have been associated with PSC-UC pathogenesis, the precise molecular factors that regulate the phenotype of this disease subtype remain unknown.

Methods

Here, we applied methyl-capture sequencing and mRNA sequencing to colonic mucosal biopsies to identify transcriptomic and epigenetic alterations differentiating UC from PSC-UC. Samples were taken from 3 groups of treatment-naïve children at diagnosis who participated in the DOCHAS study (GEN-193/11) - UC (n=10), PSC-UC (n=10) and healthy controls(n=10).

Results

Differential gene expression between UC and PSC-UC identified disease-associated genes that were either up- or down-regulated in UC or PSC relative to controls. Furthermore, expression of these genes was intricately regulated by master transcriptional regulators (pro-caspases, IL7RA) and transcription factors (AR, p53, JUND, CEBPA). Importantly, gene expression comparison between UC vs PSC-UC revealed 4 up-regulated genes in PSC-UC (RAB31, TENM3, KLHL17 and COL7A1). Notably, RAB31 and TENM3 are also significantly up-regulated in gastrointestinal (GI) cancers. We also identified 4 down-regulated genes in PSC-UC (H3C15, SLC37A2, SLC14A2 and IL20RA).

Differential methylation analysis between healthy control biopsies vs PSC-UC and UC demonstrated >1000 differentially methylated regions (DMRs, 5Kb), with majority of these sites displaying hypermethylation. Interestingly, PSC-UC vs UC analysis identified 53 regions and 59 DMRs that are hyper- and hypomethylated respectively in PSC-UC, with a large proportion of these DMRs located in gene promoters and putative enhancer regions and notably impacted gene expression of differentially expressed genes identified from the mRNAseq analysis.

Conclusion

Taken together, our study for the first time identifies distinct gene expression and DNA methylation alterations that differentiate UC from PSC-UC at diagnosis in treatment-naïve paediatric patients, some of which are associated with GI cancers. Their potential utility as predictive biomarkers of PSC development in UC or dysplasia risk in PSC-UC warrants further validation through larger patient cohorts.
DOP88 Understanding the molecular mechanisms of anti-TNF treatment failure: Whole blood DNA methylation changes associated with primary non-response to anti-TNF treatment in patients with Crohn’s disease
Authors: Lin S , Hannon E , Waring J F , Reppell M , Smaoui N , Pivorunas V L , Guay H , Chanchlani N , Bewshea C , Bai B Y H , Kennedy N A , Goodhand J R , Mill J , Ahmad T
Published: January 2023

Background

Anti-TNF treatment failure in patients with IBD is common and frequently related to low drug concentrations. DNA methylation sites from whole blood have been identified as effective biomarkers predicting treatment response to methotrexate and etanercept in patients with rheumatoid arthritis. We sought to define differences in DNA methylation associated with primary non-response (PNR) to anti-TNF treatment in patients with Crohn’s disease.

Methods

The Personalised Anti-TNF Therapy in Crohn’s disease (PANTS) study is a prospective UK-wide study investigating anti-TNF treatment failure in patients with active luminal Crohn’s disease. DNA methylation from whole blood was assessed using the Illumina EPIC Beadchip at baseline, weeks 14, 30 and 52. We compared DNA methylation profiles in a subset of adalimumab- and infliximab-treated patients who experienced PNR at week 14 and were not in remission at week 54 (infliximab = 99, adalimumab = 94) with patients who responded at week 14 and were in remission at week 54 (infliximab = 99, adalimumab = 93).

Low anti-TNF drug concentration was defined as an infliximab level <3mg/L and adalimumab level <5mg/L.

Epigenome-wide association (EWAS) analysis was performed using linear mixed models, where p values < 9 x 10-8 were considered significant. Because we observed significant changes in CD4 and CD8 T cells, B cells, NK cells, monocytes and granulocytes following anti-TNF treatment we adjusted our models for derived cell proportions (Figure 1).

Results

Overall, between baseline and week 14, we observed 4999 differentially methylated probes (DMPs) annotated to 2376 genes. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses (Figure 2).

EWAS analysis at baseline identified 48 DMPs annotated to 36 genes associated with PNR (Figure 3). The majority of DMPs (40/48) were hypermethylated (p < 0.001): of those annotated to genes, 25 were located in the gene body, 5 in the 5’ untranslated regions (UTR), 4 in transcription start sites and the remaining in 3’UTR. When EWAS analysis at baseline was stratified by anti-TNF drug concentration, 27 DMPs annotated to 17 genes were associated with PNR and low drug concentration. Of these, 11 DMPs were independently associated with low anti-TNF drug concentration at week 14 (Figure 4). We did not identify any DMPs associated with PNR with adequate drug concentration.

Conclusion

Our data suggests that baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14.
P003 MicroRNAs as biomarkers in Inflammatory Bowel Disease unclassified (IBDU) patient samples may predict the development from IBDU to Crohn’s Disease or Ulcerative Colitis
Authors: Pallikkunnath James J , Riis L B , Malham M , Høgdall E , Langholz E , Nielsen B S
Published: January 2023

Background

Differential diagnosis in Inflammatory Bowel Disease (IBD) is challenging; typically, discriminating Crohn’s Disease (CD) from Ulcerative Colitis (UC) among IBD unclassified (IBDU) patients is a diagnostic encounter in cases where the inflammatory lesions are restricted to the colon. MicroRNAs are found to be involved in the pathogenesis and are also evaluated as diagnostic biomarkers and therapeutic targets in IBD.

Methods

We obtained formalin fixed paraffin embedded (FFPE) colonic mucosal biopsies of 35 IBDU patients (including adults and children), and 8 IBD (4 CD and 4 UC) patients who were classified into CD or UC collected from the diagnostic endoscopy. Based on the patient charts, The IBDU samples were reclassified into CDs or UC or remained as IBDU after minimum 3 years clinical follow up, ie. 12 IBDU patients were reclassified as CD (IBDU-CD) and 15 IBDU patients as UC (IBDU-UC). Eight IBDU patients remained as IBDU. MicroRNA profiling was performed on all samples using Affymatrix GeneChip miRNA Arrays. Arrays were processed using pd.mirna.4.0 and oligo packages in R and were analysed using LIMMA package.

Results

We found that miR-3176 and miR-182-5p were upregulated in IBDU-CD and miR-671-5p, miR-668-5p, miR-4793-3p, and mir-6776 were upregulated in IBDU-UC with p values of less than 0.05 and 1< log [fold change] <-1 for all 6 microRNAs. Higher mucosal expression levels of miR-3176 and miR-182-5p in IBDU-CD samples compared to IBDU-UC samples, resulted in an area under the curve (AUC) of 0.87 (95% confidence interval (CI), 0.71 – 1.00) and 0.81 (CI, 0.63 – 1.00), respectively, in discriminating IBDU-CD from IBDU-UC. On the other hand, miR-3176 and miR-182-5p had AUCs of 0.69 (CI, 0.25-1.00) and 0.69 (CI, 0.21-1.00), respectively, in discriminating CD and UC. MicroRNA miR-671-5p had a higher AUC of 0.81 (CI, 0.48 - 1.00) for discriminating CDs and UCs, whereas in discriminating IBDU-CD and IBDU-UC, the AUC was only 0.64 (CI, 0.42 – 0.87).

Conclusion

We conclude that differential microRNA expression among IBDU patients can predict the development of IBDU to CD and IBDU to UC, and that the microRNA expression profile of IBDU patients differ from patients classified as CD or UC at time of diagnosis. Our findings suggest that IBDUs may have different pathogenic mechanisms for IBDU-CDs and IBDU-UCs.
P007 Paradoxical effects of high and low emulsifier diets on intestinal permeability in unstressed and stressed states in healthy human adults: a pilot randomised controlled feeding trial.
Authors: Fitzpatrick J , Gibson P R , Taylor K M , Halmos E P
Published: January 2023

Background

In preclinical models, high doses of specific emulsifiers induce intestinal inflammation. How these findings translate to Crohn’s disease (CD) is unclear, but the effects of dietary emulsifiers in the context of our food supply are needed before safe evaluation in a CD population.

Aims

To develop novel trial healthy diets matched for all dietary components, except emulsifiers; and to evaluate their suitability for study in patients with CD by defining, in healthy subjects, tolerance, blinding and adherence to the diets and their impact on intestinal permeability with and without stress.

Methods

In a pilot single-blinded, cross-over, randomised study, healthy subjects were fed for 4 weeks with ≥3-week washout between diets. Acute stress was induced via 100 mg intravenous corticotrophin-releasing hormone (CRH) on day 28 of each diet. Intestinal permeability was analysed directly with urinary lactulose to rhamnose ratio (LRR) before and at 2 weeks; fasting serum biomarkers reflecting barrier function (intestinal fatty acid binding protein, lipopolysaccharide binding protein [LBP], soluble CD14 and syndecan-1) were measured before and during each diet. All markers were also measured 2 h post CRH. Tolerance was assessed by gastrointestinal symptoms daily using a 100-mm visual analogue scale. Success of blinding was assessed by direct questioning and adherence (defined >95% consumption of only food provided) with food diaries.

Results

18 of 20 participants enrolled [median age: 34 IQR 23-46 y; 75% female] finished both diets. The diets were well tolerated with minimal overall symptoms (low: mean 9.9 (SD 8.3) vs high: 9.5 [5.5] mm). Adherence was excellent (>95%). Blinding was reasonable with 30% of participants either not knowing or choosing a high emulsifier diet as the therapeutic diet. In the unstressed state (Fig 1), LRR reduced on the HIGH diet compared to baseline (p=0.016), but no difference between diets was seen. LPB concentrations were lower in the HIGH than LOW diet (p=0.039). Under acute stress (Fig 2), LRR increased by 49% during the HIGH diet (p=0.034), but fell by 41% during the LOW diet (p=0.027). The paired CRH-induced LRR was reduced on LOW vs HIGH diet in acute stress (p=0.043). No difference in concentrations of other biomarkers were seen between diets.

Conclusion

Novel low and high emulsifier trial diets were well tolerated, achieved good adherence and reasonable blinding. Paradoxically, intestinal permeability reduced with the HIGH diet in the unstressed state, but the LOW diet protected from stress-induced increase in permeability. These diets are suitable to evaluate the effects of emulsifiers on intestinal permeability and inflammation in patients with CD.
P013 Role of IL-36RA mutations in Crohn’s disease
Authors: Hecker J , Saurenbach J , Letizia M , Löscher B S , Franke A , Kühl A A , Atreya R , Koop K , Neufert C , Trajanoski Z , Schütz A , Siegmund B , Weidinger C
Published: January 2023

Background

The interleukin 36 (IL-36) family consists of three agonists (IL-36α, IL-36β, IL-36γ), that activate the NF-κB pathway and lead to the production of pro-inflammatory cytokines and the natural antagonist (IL-36RA), which inhibits IL-36 signaling. While it is known that IL-36 signaling is important in the skin, it has been only recently reported that IL-36 might play an important role in maintaining gut homeostasis and in the development of fibrosis (Scheibe et al., 2019).

Here, we aim to understand how IL-36RA mutations contribute to development and maintenance of intestinal inflammation in Crohn’s disease.

Methods

By whole-exome sequencing (WES) and subsequently targeted Sanger sequencing we identified a Crohn’s disease (CD) patient with a heterozygous missense mutation in IL-36RA (p.Ser113Leu). To understand how the mutation affects immune cells composition and function, we characterized peripheral blood mononuclear cells (PBMCs) of the patient by mass cytometry and measured cytokine levels in the serum by ELISA.

In an available WES dataset of 177 CD patients, 47 ulcerative colitis (UC) patients and 34 healthy donors (HD), we searched for additional patients with IL-36RA mutations.

Finally, we overexpressed the identified IL-36RA mutations in HEK cells and produced mutated recombinant proteins to study the effect of the mutations on protein expression and function.

Results

Our IL-36RA mutation-carrying patient presented with a severe course of Crohn’s disease and consecutive of fibrosis, which was resistant to all commonly used treatment options. In the patient, we detected increased levels of IL-36RA, IL-18 and IL-23 in the serum and an increased frequency of TH17 cells in the blood. These findings could hint towards an over-activation of the IL-36 signaling pathway in the IL-36RA mutated patient.

In the available WES data, we identified two more CD patients with IL-36RA mutations (p.Leu133Ile, p.Pro76Leu), indicating that IL-36RA mutations are enriched in CD patients.

By overexpressing the three identified mutations in HEK cells, we could demonstrate that two mutations (p.Ser113Leu and p.Pro76Leu) lead to a reduced protein expression. Also, our current data shows that the p.Ser113Leu mutant has a reduced capacity to antagonize IL-36 stimulation. However, further testing is required for the two other IL-36RA mutants.

Conclusion

Our data suggests that IL-36RA mutations are enriched in Crohn’s disease patients and should be considered as a possible cause for therapy-refractory and fibrotic Crohn’s disease.
P020 Characterisation of mucus in patients with Crohn’s disease
Authors: Kramer C , Ziegler J , Alzain N , Schroeter S , Almalla A , Elomaa L , Addante A , Kuppe A , Fentker K , Nazat Martinez Medina J , Jarquin-Diaz V H , Rulff H , Gradzielski M , Forslund S , Mertins P , Mall M , Weinhart M , Glauben R , Siegmund B
Published: January 2023

Background

Crohn’s disease (CD) is a chronic inflammatory condition that can affect all parts of the intestine. Commonly, there is inflammation in the terminal ileum. An intestinal barrier consisting of epithelial cells covered by a mucus layer is on the one hand preventing pathogen invasion while on the other hand allowing the uptake of nutrients. However, despite the significance of the terminal ileum in CD, little is known about the mucus composition and viscoelastic properties in the terminal ileum.

In our study, we wanted to do a comprehensive analysis of the mucus in the terminal ileum of CD patients compared to healthy controls. Thus, we aspire to gain a better understanding of the role of mucus and biophysical and biochemical triggers contained in the mucus layer during the course of ileal inflammation.

Methods

During colonoscopy, native mucus and saline washes containing mucus as well as biopsies from the terminal ileum of 33 CD patients and 25 healthy controls were collected. The disease course and dietary habits of all patients and healthy controls were recorded. Samples were treated according to protocol for different types of analysis such as rheology, proteomics, metabolomics, and microbiome. Furthermore, human intestinal organoids were generated from the biopsies. Additionally, we analysed bulk RNA sequencing data from intestinal mucosal biopsies of a cohort of 128 patients, consisting of non-IBD individuals and CD patients.

Results

We here present preliminary data for the different types of analyses. Regarding the viscoelastic properties, preliminary rheology data suggests that there is no major difference between CD patients and healthy controls. As for proteomics, the first cohort is currently being analysed. Concerning the microbiome, we could show that saline washes containing mucus are more suitable than biopsies for analysing bacterial as well as host DNA. Although not statistically significant, healthy controls showed a tendency for higher alpha- diversity in microbiota. Also there were some differences in the taxonomy. Intestinal organoids were grown as cultures. In the end, the results of the analyses mentioned above will be interpreted together with the clinical data. Concerning the gene expression, we found that 12 mucin and mucin-like genes were expressed both in colon and ileum tissue. Interestingly, a subset of CD patients showed a de-novo expression of MUC5AC, MUC6, MUCL1 and MUCL3 in the ileum.

Conclusion

All in all, in our multidisciplinary study we hope to achieve a thorough analysis of the mucus layer in the terminal ileum, thus gaining new insight into the pathogenetic mechanisms in intestinal inflammation that could prospectively be used for therapeutic means.
P022 A correlation of serum Fibroblast Growth Factor 21 level with inflammatory markers and indicators of nutritional status in patients with Inflammatory Bowel Disease
Authors: Łukawska A , Mulak A
Published: January 2023

Background

Fibroblast Growth Factor 21 (FGF21) is an endocrine hormone produced mostly in the liver, adipose tissue, and the pancreas. Main inducers of the FGF21 secretion include fasting state, overfeeding, inflammation, and physical activity. FGF21 is involved in the metabolism of lipids, carbohydrates, and proteins. It also participates in body weight regulation. Previous studies have shown that serum FGF21 level is increased in animal model of colitis and patients with Inflammatory Bowel Disease (IBD) (Liu et al. Gastroenterol Res Pract 2017). The aim of this study was to assess the correlation between serum FGF21 level and inflammatory markers as well as indicators of nutritional status in IBD patients.

Methods

Fasting serum FGF21 level was measured using ELISA test in 67 patients with IBD (38 patients with Crohn’s disease and 29 patients with ulcerative colitis) and 10 control subjects. Among IBD patients, 24 subjects were in remission and 43 in acute phase of the disease. Fecal calprotectin, serum CRP, albumin and cholesterol levels were tested in all patients. BMI was also assessed. The Spearman rank correlation coefficient (R) was calculated to evaluate associations between the variables.

Results

No significant differences were found in serum FGF21 level between four studied groups: all IBD patients, IBD patients with remission, IBD patients with exacerbation and healthy controls. However, a positive correlation was observed in IBD patients between serum FGF21 and fecal calprotectin levels (R= 0.39, p= 0.003). There was also a trend toward a positive correlation between FGF21 and CRP levels (R= 0.24, p= 0.053). Moreover, an inverse correlation was found in IBD patients between FGF21 level and nutritional status indicators: BMI (R= –0.36, p= 0.003), albumin level (R= –0.38, p= 0.001), and cholesterol level (R= –0.30, p= 0.014).

Conclusion

The intensity of intestinal inflammation is related to FGF21 level, which correlates negatively with nutritional status indicators in the course of IBD. The disturbances in FGF21 secretion may contribute to the multifactorial pathogenesis of malnutrition and weight loss in IBD patients.
P028 Transitioning patients from intravenous to subcutaneous infliximab and vedolizumab for inflammatory bowel disease – what is the opportunity cost of improving access to health care?
Authors: Hilley P , Wong D , Ma R , Peterson A , De Cruz P
Published: January 2023

Background

Biologic drugs are highly effective for inflammatory bowel disease (IBD) management but are key drivers of costs of care especially when administered intravenously (IV). Availability of subcutaneous (SC) formulations has increased convenience for patients and improved access to care but at the cost of revenue to health services.

The aim of this study was to evaluate the economic impact of transitioning a tertiary centre IBD cohort from IV to SC biologic administration and assess the implications for key stakeholders.

Methods

A retrospective analysis of all patients that received IV infliximab or vedolizumab in the outpatient infusion centre of a tertiary, IBD centre between July 2019 and June 2021 was undertaken. Data were extracted from electronic medical records, pharmacy dispensing systems and the hospital business intelligence unit. An economic analysis and theoretical financial/capacity impact analysis of a transition to a SC model was estimated under two theoretical scenarios, using a random 10% and 30% of the patient cohort.

Results

Transitioning our IBD cohort from IV to SC administration would result in a loss of $2,732,123.75, comprised of $1,463,003.75 in Weighted Inlier Equivalent Separation (WIES) and $1,269,120 in drug procurement revenue. However, it would ease capacity in the infusion centre by up to 5256 hours.

Conclusion

Transitioning patients to SC administration results in improved access to infusion centres and substantial savings to state governments; however, switching results in a loss of IV-biologic generated WIES to health services. Alternative funding models are required to achieve sustainability in IBD care and reduce reliance on IV biologic generated income.
P030 Assessing dietetic interventions in inflammatory bowel disease patients
Authors: Norman E , Hoque S
Published: January 2023

Background

Nutrition can play a role in the management of inflammatory bowel disease (IBD) as well as general nutritional status, however there are many types of dietetic interventions depending on the circumstances. We decided to survey some of the nutrition interventions that IBD patients may be provided with to obtain a general picture of how helpful this is to patients, as well as the effectiveness of having a specific IBD dietetic post.

Methods

The inclusion criteria were patients with a diagnosis of IBD, reviewed by the IBD dietitian and patients had to be under the IBD team at Whipps Cross Hospital. Patients were asked if they consented to providing their email address for the anonymous survey link to be sent to. The survey was then emailed to 50 IBD patients. Nine questions were asked on the survey and an internet survey provider was used.

Results

The total response rate was 32% to the survey. The mean age was about 40 years old. 40% of patients were male with the remainder being female. 58% of the patients had Crohn’s disease with the remainder having UC. 100% of patients felt the dietitian involved them in decision making. The results of the following dietetic interventions are only where applicable to patients: 100% of patients rated nutrition support care (including exclusive enteral nutrition for Crohn’s disease) as excellent or good. 100% of patients rated specific IBD dietary advice as excellent or good. 100% of patients rated functional symptoms or irritable bowel syndrome advice as excellent or good. 100% of patients rated artificial nutrition advice as good. 81% of patients felt advice improved symptoms and nutritional state with 13% being unsure. 81% of patients felt advice improved quality of life with 13% being unsure. 81% of patients felt cultural, lifestyle and language needs were considered with 13% being unsure. 100% of patients felt having access to a dietitian was helpful.

Conclusion

All patients felt that the specific types of dietary care provided were excellent or good, with the majority of patients responding that advice improved symptoms, improved quality of life and that cultural and lifestyle needs were taken into account. All patients found it supportive to have a dietitian as part of the IBD multidisciplinary team. It would be useful in the future to further assess these types of dietetic care, such as exclusive enteral nutrition for Crohn’s disease. These findings support the best practice guidelines for having a dietetic post as part of the IBD team.
P031 CKD-506, a new histone deacetylase 6 inhibitor, suppresses immune cells and restores intestinal epithelial function
Authors: Lee J Y , Ma H W , Kim J H , Park I S , Son M , Ryu K H , Shin J , Kim W H , Kim S W , Cheon J H
Published: January 2023

Background

Inflammatory bowel disease (IBD) represents a group of chronic immune-mediated diseases of the gastrointestinal tract, characterized by recurrent inflammation and consequential damage of the gastrointestinal tract. Histone deacetylases (HDACs) are a family of mostly ubiquitous enzymes that remove acetyl groups from lysines on histone proteins to regulate gene transcription. HDAC6, which is localized to the cytoplasm, appears to be a promising candidate in IBD treatment. The purpose of the current study was to examine the anti-inflammatory effects of CKD-506, a novel histone deacetylase 6 (HDAC6) inhibitor, on human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells and to explore the relationship between CKD-506 and gut epithelial barrier function.

Methods

Lipopolysaccharide (LPS)-stimulated human PBMCs from inflammatory bowel disease (IBD) patients were treated with CKD-506, and tumor necrosis factor-alpha (TNF-α) expression was measured using enzyme-linked immunosorbent assay. The proliferation of CD4+ T cells from IBD patients was evaluated using flow cytometric analysis. The effects of CKD-506 on gut barrier function in a cell line and colon organoids, with examinations of mRNA production, goblet cell differentiation, and E-cadherin recovery, were investigated using quantitative reverse transcription PCR, immunofluorescence, and FITC-dextran permeability assay.

Results

TNF-α secretion, a pivotal pro-inflammatory mediator in IBD, of LPS-triggered PBMCs was markedly decreased by CKD-506 treatment in a dose-dependent manner and to a greater extent than by tofacitinib or tubastatin A treatment. E-cadherin mRNA expression and goblet cell differentiation increased significantly and dose-dependently in HT-29 cells in response to CKD-506, and inhibition of E-cadherin loss after TNF-α stimulation was significantly reduced both in HT-29 cells and gut organoids. Caco-2 cells treated with CKD-506 showed a significant reduction of barrier permeability in a dose-dependent manner.

Conclusion

The present study demonstrated that CKD-506 has anti-inflammatory effects on PBMCs and CD4+ T cells and improved gut barrier function, indicating its potential as a promising and competent candidate for small-molecule medicine for IBD treatment.
P032 Distinct immune cell trigger responses in Ulcerative Colitis – Results from an in vivo human intestinal injury model
Authors: Bronze M , Vilstrup Johansen J , Attauabi M , Tveiten Bjerrum J , Ramírez Galera M , Woetmann A , Benedict Seidelin J
Published: January 2023

Background

Ulcerative colitis (UC) is characterized by alternated periods of active inflammation and quiescent disease. The mechanisms behind flare-up reactions are elusive, but it is hypothesized that normally unharmful factors can initiate flares in genetically susceptible individuals with a dysfunctional mucosal barrier and hyper-responsive immune system, leading to sustained inflammation. We aimed to describe the early cellular responses to injury in the normal intestine and in UC patients in remission.

Methods

Experimental injuries were performed in six UC patients in remission (endoscopic Mayo sub-score 0 and total Mayo score below 3) and in six control subjects1. During the initial sigmoidoscopy, six experimental wounds were made. After 24 and 48 hours, the subjects were re-endoscoped and new biopsies were taken across the experimental injury by angling the biopsy forceps 90° (Figure 1), thus including the mucosa of the injury edge and the wound bed. Biopsies from each time point were sequenced (bulk RNA-sequencing) and the frequencies of different cell types were inferred using deconvolution (Cellanneal)2

Results

Type 1-conventional dendritic cells (cDC) and macrophages decreased in both UC and controls after injury (p<0.05). Inflammatory monocytes increased in controls only at 48h post-injury, while in UC, these cells increased at 24h and decreased at 48h post-injury. There were no significant changes in natural killer cells or T and B cells frequencies between UC and controls both pre- and post-injury. The frequency of ILCs, mast cells, and type 2-cDC continuously increased in UC patients from pre-injury to 48h post-injury, in contrast to controls, where they decreased (Figure 2). ILC marker CD117 (expressed by ILC3 and ILC2) was overexpressed in UC pre-injury, however both CRTH2 (expressed only by ILC2) and CD127 were similar pre- and post-injury. Finally, the cytokines required for ILC3 stimulation (interleukin (IL)-1β, IL-1α and IL-23) and IL-17A, produced by ILC3 during inflammation, were increased.

Conclusion

UC patients in complete remission show an increased innate inflammatory response to mucosal injuries compared to healthy individuals, highlighted by the early engagement of inflammatory monocytes, mast cells, and ILCs, particularly ILC3s. Our data show a UC-specific innate cellular hyper-response that may explain the initial mechanisms of flares and could be targeted in future anti-flare therapies.

References

1. Seidelin JB et al. Acute Experimental Barrier Injury Triggers Ulcerative Colitis-Specific Innate Hyperresponsiveness and Ulcerative Colitis-Type Microbiome Changes in Humans. Cell Mol Gastroenterol Hepatol 2021;12:1281-1296.

2. Buchauer L, Itzkovitz S. cellanneal: A User-Friendly Deconvolution Software for Omics Data, 2021.
P037 Enhancing the efficacy of cannabidiol using protein nanoparticles to treat inflammatory bowel disease
Authors: Moniruzzaman M , Rewatkar P , Begun J , Popat A
Published: January 2023

Background

Inflammatory bowel disease (IBD) is a global health burden and approximately 30% of the patients with severe IBD require surgery following treatments, leaving an unmet need of new and safe therapies. Cannabidiol (CBD) is one of the major non-psychoactive and water-insoluble constituents of medicinal cannabis, known for its anti-inflammatory properties in epilepsy. The compound is safe at high doses and doesn’t have the psychotropic effects of delta-9-tetrahydrocannabinol. Therefore, this study investigated the therapeutic potential of CBD in controlling intestinal inflammation.

Methods

CBD was loaded in the protein nanoparticles (PNP-CBD) and tested for its water solubility. Primary intestinal epithelial cells (IECs; organoids) and bone marrow-derived macrophages (BMDM) were isolated from C57BL/6 mice and treated with IL-1β and LPS, respectively. Stimulated cells were treated with ±CBD and checked for downstream signalling through inflammatory gene expression using qRT PCR. Conditioned media from LPS-treated BMDM was also harvested to treat mIECs to check link between macrophage activation and intestinal inflammation. Accordingly, we assessed the efficacy of CBD in suppressing intestinal inflammation using dextran sulfate sodium (DSS)-induced acute experimental colitis in the C57BL/6J mice. At the end, blood samples were collected for hematology and serum analyses, mesenteric lymph nodes for immune cell signatures, colon samples for histological analysis and qRT PCR analysis for inflammatory genes.

Results

CBD loading in the protein nanoparticles (PNP-CBD) increased its water solubility up to 60 times. In the mIECs, CBD did not have any effects on cytokine-induced inflammation; however, it significantly dampened LPS-induced BMDM activation and inflammatory cytokine production. The results also showed that inhibition of CB2 but not CB1 receptor can completely abolish the anti-inflammatory potential of CBD in the BMDM. Transferring conditioned media from BMDM to mIECs showed macrophage-specific targeting alleviated inflammation in the mIECs. These have also been observed in the acute DSS colitis model where not CBD alone but PNP-CBD improved disease severity index including body weight loss, diarrhoea, shorter colon length, and inflammatory gene expression in the colon. PNP-CBD also reduced histological colitis and increased goblet cell mucin production in the DSS-treated colon.

Conclusion

Encapsulation of CBD increased water solubility and efficacy in alleviating intestinal inflammation in ulcerative colitis. These data also demonstrated that CB2 receptor targeting of macrophage activation is the primary mechanism of CBD’s effect.
P041 The upregulation of tissue factor in colonic mesenchymal cells in active ulcerative colitis is associated with severe and resistant to treatment disease, but tofacitinib has not effect on it
Authors: Drygiannakis I , Tzenaki N , Archontoulaki E , Filidou E , Kandilogiannakis L , Valatas V , Kolios G , Koutroubakis I E
Published: January 2023

Background

Tissue factor (TF) plays an important role on blood clotting and the risk of thromboembolic events is increased in ulcerative colitis (UC). Tofacitinib, recently introduced in UC therapeutics, may further increase the risk.

To delineate thrombosis pathophysiology in this context, TF expression in primary human colonic mesenchymal cells (PHCMC) of patients with active UC was correlated with clinical parameters, serum markers of inflammation and endoscopy. We then treated those PHCMC with tofacitinib with or without cytokines to better mimic the in vivo milieu they are exposed to.

Methods

PHCMC from endoscopic biopsies of the inflamed mucosa of 10 UC patients with an endoscopic Mayo score ≥2 were treated with all major T helper (Th)1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or T regulatory (Treg; TGF-β, IL-10) cytokines with or without tofacitinib. Cells were lysed, RNA was isolated and reverse-transcribed to cDNA. TF and RPL4 (housekeeping) cDNAs were quantified with real-time PCR. Wilcoxon and Mann-Whitney U tests were used to compare TF ΔΔCT for paired or unpaired values, respectively, and Spearman’s rho to correlate TF ΔΔCT with scale clinical variables and continuous laboratory values.

Results

Increased TF mRNA abundance in PHCMC was correlated to increased partial Mayo score (Spearman’s rho 0.661, p < 0.044), reduced serum albumin (Spearman’s rho -0.723, p < 0.05) and to more severe endoscopic lesions: cells originating from UC patients of an endoscopic Mayo score 3 expressed 20 times more TF than those from an endoscopic Mayo score 2 (p < 0.017; Figure 1). Moreover, PHCMC from difficult-to-treat patients, defined as requiring >1 biologics, expressed 9 times more TF (Figure 2).

On the other hand, treatment with tofacitinib did not upregulate TF (Figure 3). PHCMC expressed receptors and responded to treatment with all major Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or Treg (TGF-β, IL-10) cytokines by further upregulating TF by 5.5-7.5 times (p 0.001-0.035). IL-13 had the maximal effect (Figure 4). Even when tofacitinib was added together with the aforementioned cytokines, it did not further upregulate TF; instead, it tended to partially inhibit their effects. For example, it decreased upregulation of TF by IL-13 by 40%.

Conclusion

The expression of TF mRNA in active UC is significantly associated with clinically and endoscopically severe disease and resistance to treatment. Tofacitinib per se does not increase TF. Instead, it may limit the upregulating effect of pro-inflammatory cytokines.
P042 Distinct gene expression profile associated to inflammation and neoplastic progression in longstanding ulcerative colitis
Authors: Alsøe L , Lefol Y , Wennerström A B , Liu Y , Andersen S N , Klepp P , Brackmann S , Nilsen H
Published: January 2023

Background

One of the main drivers of colitis associated colorectal cancer (CA-CRC) is suspected to be long-standing mucosal inflammation, but far from all colitis patients develop cancer. We analyzed mucosal gene expression profiles in ulcerative colitis (UC) patients who have progressed to neoplasia (progressors) compared to those who have not (non-progressors) in order to identify underlying mechanisms for neoplastic progression in UC patients.

Methods

We performed transcriptome profiling on 143 mucosal samples from non-neoplastic colon segments of 14 UC progressors and 30 UC non-progressors from the prospective CA-CRC cohorts from Lovisenberg Diakonale- and Akershus University Hospitals in Norway. Differentially expressed genes were determined using SurePrint G3 human gene expression 60K microarray (Agilent Tech Inc.).

Results

609 genes were differentially expressed between non-progressor- and progressor inflamed tissue, 24 genes between non-progressor- and progressor non-inflamed tissue, 404 genes between non-progressor non-inflamed- and -inflamed tissue and 26 genes between progressor non-inflamed- and inflamed tissue.

In inflamed mucosa, genes related to metabolic processes such as xenobiotic- as well as carbohydrate and lipid metabolism were significantly higher expressed in progressors compared to non-progressors. Genes related to innate and adaptive immune responses were expressed at significantly lower levels. These main traits were not influenced by type of inflammation but apparent in both, chronic- and chronic active inflammation. However, chronic inflamed mucosa of progressors were also characterised by dysregulation of processes related to chromatin organization. Treatment with immunomodulators and biologics were equally distributed in the groups. Restriction of the analysis to patients without treatment of these drugs did not significantly influence the main results.

In non-inflamed mucosa, the gene expression profile of progressors and non-progressors were remarkably similar. In fact, within the positively and negatively enriched gene ontologies, only the gene S100P was significantly deregulated with a higher expression in non-inflamed mucosa of the progressors.

Conclusion

Upregulated detoxification as well as dysregulated immune response and chromatin organization may be underlying processes for neoplastic progression in ulcerative colitis. Our findings warrant further evaluation.
P043 ALI culture derived from human organoids recapitulates cell composition during intestinal crypt formation
Authors: Dotti I , Álvarez I , Esteller M , Ricart E , Salas A
Published: January 2023

Background

The ALI (air liquid-interface) culture derived from human primary intestinal epithelium is generating considerable interest for closely reproducing the mature cell features of the differentiated epithelium in a 2D configuration. However, the phenotypic changes occurring in the human ALI culture during cell differentiation have thus far not been explored in depth. Here, we aimed to characterize the cell composition of human ALI culture over time.

Methods

3D epithelial organoids (n=3) were expanded from human colonic samples and organoid-derived single cells were seeded on a Transwell. After reaching confluence, the submerged 2D was switched to ALI and maintained for 7 days. The ALI culture was analysed every 24h by qPCR, membrane staining (H&E and immunofluorescence) and trans-epithelial resistance (TER) (Figure 1).

Results

The confluent pre-ALI (TER>200 Ω*cm2) showed high mRNA expression of stem cell markers (i.e., LGR5, AXIN2), while several differentiation markers (i.e., ZG16, CLCA1, ANPEP, AQP8, BEST4, CHGA) were almost undetectable, thus resembling the stem cell niche compartment. Air exposure of the 2D culture induced an early and transient (limited to ALI-day 1) transcriptional up-regulation of proliferation markers (i.e., MKI67, CDCA7) and OLFM4, followed by a concomitant marked downregulation of stem cell markers, thus mimicking the Transit Amplifying compartment. OLFM4 protein, as observed by immunofluorescence, showed an early cytoplasmic accumulation followed by secretion at later time points (from ALI-day 5). Differentiation towards absorptive and secretory lineages increased gradually from ALI-day 5 and ALI-day 7 onward, respectively, at both the transcriptional and protein level. Expression changes were accompanied by the progressive transition of the ALI monolayer from a squamous to a columnar morphology. In parallel, TER promptly increased upon ALI switch, thus indicating the early establishment of cell-cell junctions.

Conclusion

By recapitulating the cell composition identified along the crypt, the ALI culture holds promise to be a versatile in vitro model for studying how the different epithelial cell compartments respond to specific environmental stimuli, like those present during an inflammatory flare.
P048 Deep phenotyping of the peripheral immune cell compartment in Crohn‘s Disease
Authors: Haag L M , Walling S , Kunkel D , Hecker J , Letizia M , Huck A , Weidinger C , Glauben R , Kuehl A , Siegmund B
Published: January 2023

Background

Crohn’s disease (CD) is characterized by chronic inflammation that can discontinuously affect any segment of the gastrointestinal tract. New findings concerning clinical behaviour, epidemiology, genetics, and gut microbiota suggest that CD in the ileum (iCD) and the colon (cCD) should be considered as two distinct subtypes of IBD. As systemic immune cell signatures differ between CD and UC, we propose a similar strategy for the differentiation between iCD and cCD. The aim of this study is to analyse and compare peripheral blood mononuclear cell (PBMC) subsets of iCD and cCD patients. For a robust analysis of the peripheral immune compartment in inflammatory bowel diseases (IBD), PBMC subsets of ileocolonic CD (icCD), ulcerative colitis (UC) and healthy controls (HC) were included as well.

Methods

In this study a total of 50 adults, 10 subjects per subgroup – iCD, cCD, icCD, UC and HC – were included. From each individual, both naïve and PMA/Ionomycin stimulated samples were stained for phenotypic and functional characterization using mass cytometry. For data analysis pre-gating was performed in Cytobank followed by debarcoding, compensation, normalization, clustering, and analysis on differential abundances edge R (DA_edgeR) and differential states limma (DS_limma) with CATALYST in R.

Results

FlowSOM allowed us to cluster CD45+ PBMCs of all samples into 16 major cell subtypes as visualized via UMAP (Fig. 1). DA testing showed significant differences between iCD and cCD, iCD and icCD but not cCD and icCD (Fig 2). Significantly different abundances among CD subgroups and HC included cluster 1 CD33+ myeloid cells, 7 CD45RO+ CD16+ cells, 8 CD11b+ cells, 9 CD4+ IL7R+ T cells and 12 CD27+ CD38+ CD45RA+ B cells (Fig. 3).

When focussing on differences between iCD, cCD and icCD, DS testing revealed significantly higher IFNgamma and TNFalpha production in cluster 9 (CD4+ IL7R+ T cells), 15 (CD8+ T cells) and 16 (CD8+ CD45RO+ T cells) in iCD compared to icCD. Moreover, cluster 9 showed significantly higher levels of IFNgamma in iCD compared to cCD, and for TNFalpha cluster 16 revealed significantly higher levels in cCD compared to icCD (Fig. 4).

Fig. 1 UMAP clustering of stimulated cells.

Fig. 2 Differential analysis of stimulated samples with DA_edgeR and DS_limma.

Fig. 3 Relative population abundances with DA_edge R testing.

Fig. 4 Relative state marker abundance within clusters with DS_limma testing.

Conclusion

Deep phenotyping of the peripheral immune cell compartment in CD might serve as a confirmatory marker for disease location and therefore as predictor of clinical behaviour.