bluuebunny/crossref_metadata_2025 huggingface dataset:
P005 The solute carrier LC22A4/Organic Cation Transporter (OCTN)-1 as a novel Inflammatory Bowel Disease determinant at the microbe-host interface
Authors: Masi L , Petito V , Fidaleo M , Palucci I , Del Chierico F , Ivagnes V , Mercuri A , Graziani C , Lopetuso L R , Armuzzi A , Gasbarrini A , Pani G , Scaldaferri F
Published: January 2022

Background

The putative Ergothioneine Transporter, SLC22A4/OCTN1, is expressed in the intestine and monocytes/macrophages, and its widely represented missense variant L503F (0.4 Minor Allele Frequency in Caucasians) has been found to be associated with Crohn’s disease, sporadic colorectal cancer in early age and ulcerative colitis (UC) patients. The leucine-to-phenylalanine substitution at amino acid 503 affects OCTN1 capacity to transport ergothioneine, acetylcholine and likely other substrates of endogenous or microbial origin; however, whether this defect contributes to inflammatory bowel disease (IBD) pathogenesis, and the mechanistic underpinnings, remain elusive.

Methods

In order to gain mechanistic insight on the role of SLC22A4 in IBD, we first analyzed primary monocytes from healthy donors and UC patients of varying OCTN1 genotypes. Then we stably knocked down or overexpressed OCTN1 and its disease-associated variant in the monocytic cell line THP-1. Finally, we compared wild-type and OCTN1 knockout C57BL/6 mice in a chemical colitis paradigm in vivo.

Results

Primary adherent monocytes from healthy donors homozygous for the IBD-associated allele (TT) displayed a significant enhancement of interleukin 1 beta (IL-1β) response to peptidoglycan (PGN) compared to the other genotypes. Likewise, cells from UC patients bearing the 503F variant released more IL-1β in response to live bacteria and displayed reduced expression of autophagy markers (e.g. p62, LC3), compared to patients negative for the variant.

In agreement with these findings, OCTN1-deficient THP-1 macrophages secreted reduced amounts of IL-1β when challenged in vitro with PGN or live bacteria. Most relevant, THP1 cells engineered to overexpress the 503F variant of OCTN1 displayed a much stronger IL-1β response to muramyl dipeptide and PGN challenge, compared to cells transduced with the “wild type” transporter. Also consistent with a role for OCTN1 variants in inflammatory cascades relevant to IBD, in vivo studies revealed milder DSS-induced colitis in OCTN1 knockout mice compared to wild-type animals, both at the peak of disease severity and at the end of the recovery period.

Conclusion

Collectively our initial evidence supports the view that OCTN1 may have a causative role in IBD via deranged bacterial sensing and cytokine production, with 503F likely behaving as a gain-of-function variant. Thus, targeting OCTN1 variant may provide unprecedented opportunities for the personalized treatment of IBD patients and a better understanding of disease pathogenesis.
P023 Prevalence of malnutrition, risk of malnutrition and quality of life among patients with Inflammatory Bowel Disease
Authors: Korwel K M , Hjortswang H , Eberhardson M , Pihl Lesnovska K
Published: January 2022

Background

The relapsing and remitting course of inflammatory bowel disease (IBD) with inflammation and gut symptoms may result in an elevated risk of malnutrition and impaired quality of life. Patients with pro-longed active IBD are at the highest risk of developing impaired nutritional status. The aim of this study was to determine the prevalence of malnutrition and risk of malnutrition in outpatients with inflammatory bowel disease. Furthermore, to study their body composition, identify factors associated with malnutrition and investigate the association between malnutrition and quality of life.

Methods

A total of 328 IBD outpatients, (154 [47 %] with Crohn’s disease (CD) and 174 [53%] with ulcerative colitis (UC)) at Linköping University hospital were included in this population-based cross-sectional study. Patients underwent malnutrition assessment using the recently published Global Leadership Initiative on Malnutrition (GLIM) criteria. Data about body mass index, weight loss and dietary problems were collected and used to evaluate risk of malnutrition. The Short Health Scale (SHS) was used to measure quality of life.

Results

Malnutrition was detected in 33 patients (10,1 %) and 162 patients (55,0 %) had an increased risk of malnutrition. Malnutrition was associated with lower quality of life in the entire patient cohort and there was no difference between diagnoses. The prevalence of malnutrition was significantly higher among women (73% vs 27 %). Malnourished patients and those at risk of impaired nutritional status were more likely to have had active disease during the preceding 12 months compared to patients without malnutrition or at risk of malnutrition (27 % vs 12% [p=0.015] and 15 % vs 7% [p=0.037] respectively). Surprisingly, a higher calprotectin value was detected among patients without risk of malnutrition when compared to those who were at risk (median 46 [IQR 17 – 106] vs 59 [IQR 25 – 288] respectively, p=0,009). Women with IBD had significantly lower fat-free mass index (FFMI) compared to men. Patients with CD had reduced quality of life compared to patients with UC.

Conclusion

The prevalence of malnutrition in IBD-outpatients was 10% and more than half of the participants were at risk of malnutrition. Malnutrition was associated with poor quality of life. Malnutrition screening might be important in this population as ongoing inflammation and active disease seem to be apparent even among patients without clear nutrition difficulties or weight problems, but might influence nutritional status and quality of life negatively in the long term. Patients with IBD should therefore be screened for malnutrition and risk of malnutrition on a regular basis.
P024 CD40 and key CD40/CD154 pathway members are significantly upregulated in inflamed lesional IBD biopsies as shown by whole transcriptome AmpliSeq gene expression profiling and corresponding immunohistochemistry
Authors: Raulf F , Delucis-Bronn C , Angevaare E , Caesar N , Marcantonio A , Ceci M , Regnier C , Bonal C , Siegel R , Niess J H , Wieczorek G , Hrúz P
Published: January 2022

Background

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with growing evidence of a critical role of the CD40/CD40L pathway (Shenhaji et al Front Immunol 6:529). To confirm and extend these findings, we profiled colonic biopsies from IBD patients with active ulcerative colitis (UC) and Crohn’s disease (CD) as well as non-IBD controls (HV).

Methods

Mucosal inflamed and non-inflamed biopsies of IBD patients and of healthy control subjects were immediately immersed in RNAlater (for RNA) or formaldehyde (for histology). RNA was extracted by RNeasy (Qiagen) with DNase digestion, quantified by UV spectrophotometry, and quality-controlled by Bioanalyzer (Agilent). 10 ng total RNA was subjected to ultrahigh-multiplexed RT-PCR using the AmpliSeq Transcriptome Human Gene Expression kit for 20,802 genes (Thermo Fisher). Barcoded samples were multiplexed and amplicons sequenced on an Ion GeneStudio S5 (Thermo Fisher). The resulting Excel matrix of normalized reads [RPM = Reads Per Million mapped reads; equivalent to TPM, Transcripts Per Million] was analyzed by Qlucore Omics Explorer 3.7 (Qlucore) and GraphPad Prism 9 (GraphPad Software). For histology, 2 μm sections of FFPE colonic biopsies were subjected to standard hematoxylin and eosin stain, and to automated immunohistochemistry (IHC; Ventana Discovery XT & Leica BOND RX) using standard cell-type specific antibodies and anti-CD40 Ab213205 (Abcam) and anti-CD40L AF617 (R&D Systems).

Results

161 colonic biopsies were analyzed from 36 CD and 26 UC patients (from inflamed and non-inflamed mucosa per subject), and 14 HV. Unbiased gene expression analysis by principal component analysis and hierarchical clustering showed upregulation of known inflammation markers and inflammasome components. In inflamed mucosa mRNA levels of CD40 (figure), CD154, CD44, CD86, KYNU, FCRL4, and others revealed a significant consistent upregulation of the CD40/CD154 pathway. Representative corresponding IHC stainings confirmed the upregulation of CD40 and CD154 at the protein level showing strong expression of CD40 in lesional biopsies (inflamed areas and epithelial cells), moderate expression in non-lesional and low in HV biopsies (no expression in epithelial cells), while higher expression of CD154 was observed in lesional and non-lesional biopsies compared to HV.

Conclusion

Consistent upregulation and activation of the CD40/CD154 axis was observed in inflamed mucosa of patients with IBD. Together with other (pre-)clinical evidence pointing to the importance of CD40 signaling, manipulation of this pathway might be a potential therapeutic target in IBD.
P025 A review of the newly implemented Inflammatory Bowel Disease dietetic service
Authors: Norman E
Published: January 2022

Background

Nutrition plays an important role in the onset and later the management of inflammatory bowel disease (IBD). Patients are becoming progressively interested in non-pharmacological methods of managing IBD and the dietary advice around this. It is reported that only 7% of services have enough dietitians to meet the IBD Standards recommendation. A year after the implementation of a new dietetic post at Whipps Cross Hospital, it was decided to review the effectiveness of this post for patients with a survey.

Methods

The inclusion criteria were patients with a diagnosis of IBD, reviewed at least twice by the IBD dietitian and patients who are under the IBD team at Whipps Cross Hospital. Patients were telephoned and asked if they consented to providing their email address for the anonymous survey link to be sent to. The survey was emailed to 40 patients. Six questions were asked on the survey, the survey provider was Survey Monkey.

Results

The final cohort included a 53% response rate to the survey. Age range was 18–64 years old. 95% of patients felt their consultation time was long enough. 95% of patients thought they had sufficient dietetic contact. 95% of patients felt the dietitian involved them in decision making. 67% of patients thought nutrition input improved their health/quality of life, with 29% being unsure. 100% thought having access to an IBD dietitian was useful. 71% of patients rated their dietetic care as excellent and 29% rated it as good.

Conclusion

The response from the majority of IBD patients was that nutrition input was helpful; with all patients finding the care provided by the dietitian was either good or excellent. All patients found it supportive to have dietetic access as part of a multidisciplinary approach to their IBD care. These findings support the best practice guidelines for having a dietitian post as part of the IBD team.
P028 Inhibition of stromal glycolysis by targeting PFKFB3 decreases experimental colitis
Authors: Zhou Z , Plug L G , de Jonge-Muller E S M , Abou Elmagd A , van der Meulen-de Jong A E , Barnhoorn M C , Hawinkels L J A C
Published: January 2022

Background

Studies in fibrotic diseases revealed that glycolysis is the preferred energy source for fibroblasts. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) has the highest kinase activity to shunt glucose toward glycolysis. Therefore inhibition of PFKFB3 has been proposed as a potential target for several cancers and inflammatory diseases. However, the metabolic status of fibroblasts in patients with inflammatory bowel disease (IBD) and the role of PFKFB3 are currently unknown.

Methods

Single-sample gene set enrichment analysis (ssGSEA) of GSE16879 was performed to evaluate metabolic changes in IBD. Seahorse real-time cell metabolic analysis was performed to explore the metabolic activity of fibroblasts. Next the expression of PFKFB3 in primary patient derived intestinal fibroblast was determined by quantitative PCR (qPCR) and western blot under normal and inflammatory conditions. Proliferation and migration of fibroblasts were measured using colony formation and wound healing assays. In order to evaluate the effect of the inhibition of PFKFB3 in vivo, PFK15, a specific inhibitor of PFKFB3, was intraperitoneal injected in mice with dextran sodium sulfate (DSS)-induced colitis and in the T-cell transfer model for colitis. Next to clinical parameters, the abundance of α-smooth muscle actin (α-SMA) expressing fibroblasts, immune cells (CD45) and endothelial cells (CD105) was determined by immunohistochemistry.

Results

The ssGSEA analysis revealed glycolysis was significantly higher in IBD patients, compared to healthy controls. Consistently, the expression of PFKFB3 was also elevated in a cohort of inflamed intestinal tissues from IBD patients compared to non-inflamed sites from the same patient or healthy controls. On the cellular levels, this analysis showed that PFKB3 expression was higher in IBD-derived stromal cells compared to healthy or non-inflamed stromal cells. In vitro PFKFB3 expression in fibroblasts was increased after the stimulation with pro-inflammatory cytokines like TNF-α and a mix of cytokines often upregulated in IBD patients: interleukin (IL)-17A, oncostatin M (OSM) and IL-1β. As for the metabolic changes, inflamed fibroblasts had a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reverted by inhibition of PFKFB3 using PFK15. Furthermore, PFK15 suppressed the proliferation and migration of fibroblasts. The in vivo experiments showed that PFK15 reduced the severity of the colitis, accompanied by a reduction of the total amount of immune cells (CD45), activated fibroblasts (α-SMA) and angiogenesis (CD105).

Conclusion

Increased PFKFB3 expression seems to contribute the inflammation and the pathological function of fibroblasts in IBD.
P038 Targeting the hepcidin-ferroportin axis with Nrf2 agonists to treat iron deficiency anaemia in Inflammatory Bowel Disease
Authors: Martin J , Aksan A , Steinhilber D , Ulrich-Rückert S , Stein J M
Published: January 2022

Background

Anaemia of inflammation is a common extraintestinal manifestation of IBD, impacting patient quality of life and hospitalisation rates. High circulating levels of proinflammatory cytokines induce hepatic production of the iron-regulatory peptide hormone hepcidin. By binding to the iron exporter ferroportin, hepcidin induces its internalisation and degradation, restricting the bioavailability and absorption of iron. This study aimed to test the effect of dimethyl fumarate (DMF) and sulforaphane (SFN), known activators of the transcription factor Nrf2, on the hepcidin-ferroportin (hep-fer) axis.

Methods

HepG2 cells were cultivated under standard conditions and treated with DMF or SFN for 16h. For reporter gene assays, plasmids were transfected by lipofection; luciferase activity was measured luminometrically. Quantitative real-time PCR was performed to determine mRNA expression. Proteins were detected by Western blot or LC-MS/MS. For gene knockdown, HepG2 cells were transfected with targeting or nontargeting siRNA duplexes. Graph Pad Prism v.7.00 and IBM SPSS 25 were used for statistical analysis.

Results

Both DMF and SFN reduced basic hepcidin expression and counteracted cytokine-induced hepcidin expression at promoter, mRNA and protein levels in HepG2 cells, independent of Nrf2 activation. Furthermore, DMF caused upregulation of ferroportin and ferritin at mRNA level, dependent on induction of Nrf2 and its downstream target haem oxygenase-1 (HO-1).

Conclusion

The Nrf2 agonists DMF and SFN might ameliorate anaemia of inflammation by disrupting the hep-fer axis by Nrf2-dependent and -independent mechanisms, and may be a promising therapeutic option for anaemia of inflammation for patients with IBD.
P051 IFNγ-macrophages could mediate EMT in Crohn’s disease through the WNT pathway
Authors: Edo M M , Macias-Ceja D C , Bauset C , Lis-Lopez L , Coll S , Seco-Cervera M , Cosin-Roger J , Calatayud S , Barrachina M D , Ortiz-Masiá D
Published: January 2022

Background

Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients.

Methods

The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT) through the WNT pathway. The mRNA and protein expression of IFNγ in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (10 ng/ml) for 4 days, the mRNA expression of WNT2b, WNT6 and TGFβ were determined by RT-PCR and protein. IFNγ-U937 were coculture with HT29 cells for 3 days and the expression of EMT markers, βCATENIN and WNT2b in HT29 cells were analyzed by WB. In some cases, HT29 cells were treated with the inhibitor of the WNT-pathway, XAV939 (1 μM), or were transfected with vectors-targeting human FZD4 (miFzd4). Results are expressed as mean±SEM. Statistical analysis was performed by ANOVA + Newman-Keuls or unpaired t-test.

Results

The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 70,0 ± 2,3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 80,4 ± 6,8 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 18,9 ± 0,3 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the protein expression of WNT2b (1,3 ± 0,07 N=13* vs vehicle) but not the expression of WNT6 or TGFβ. IFNγ-U937 co-cultured with HT29 increased significantly the protein expression of EMT markers, βCATENIN and WNT2b in HT29 cells (VIMENTIN: 2,7 ± 0,3 N=6* vs vehicle-HT29; SNAIL1: 1,5 ± 0,1 N=5* vs vehicle-HT29; βCATENIN: 1,4 ± 0,09 N=5* vs vehicle-HT29; WNT2b: 2,0 ± 0,3 N=8* vs vehicle-HT29). Treatment HT29 cells with XAV939 (VIMENTIN: 1,4 ± 0,4 N=6 vs vehicle-HT29 XAV; SNAIL1: 1,0 ± 0,1 N=5 vs vehicle-HT29 XAV; βCATENIN: 0,8 ± 0,1 N=5 vs vehicle-HT29 XAV) or with miFzd4 (VIMENTIN: 0,3 ± 0,07 N=12* vs IFNγ-HT29 mock; SNAIL1: 0,5 ± 0,06 N=12* vs IFNγ-HT29 mock; βCATENIN: 0,6 ± 0,04 N=12* vs IFNγ-HT29 mock; FZD4: 0,5 ± 0,04 N=12* vs IFNγ-HT29 mock) partially reversed the increases produced by IFNγ-U937 in HT29 cells.

Conclusion

IFNγ-macrophages may stimulate the expression of WNT ligands in an autocrine and paracrine manner. The expression of WNT ligands at the epithelial level could favor mesenchymal epithelial transition through WNT2b ligand and FZD4 receptor.
P055 Factors from human mesenteric adipose tissue control intestinal stem cell differentiation and proliferation in vitro
Authors: Ziegler J , Gerbeth L , Brunkhorst M F , Glauben R , Weidinger C , Siegmund B
Published: January 2022

Background

In Crohn’s disease, hyperplastic mesenteric adipose tissue wrapping around inflamed small intestinal segments is a pathognomonic finding. Factors secreted by this so-called creeping fat can influence immune cells and affect disease severity in animal models. However, data on the interactions of adipose tissue and the intestinal epithelium are still sparse and derive almost exclusively from cell lines and animal models. Therefore, we employed primary human intestinal organoid culture and primary human adipose tissue explants to elucidate the role of adipose tissue-derived factors on the epithelium.

Methods

Creeping fat and unaffected mesenteric adipose tissue from a Crohn’s disease patient was used to produce adipose tissue-conditioned medium (ATcM). ATcM or control medium was added to growth medium (contains Wnt3a) and differentiation medium (Wnt3a removal) during primary human ileum-derived organoid culture. We analysed organoid morphology, proliferation, and phenotype by light microscopy, live cell imaging, resazurin viability assay, and reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Results

In the presence of ATcM, differentiation of organoids was accompanied by a subtle and only transient delay in the development of morphologic signs of differentiation, namely organoid budding and darkness. In line, RT-qPCR at day 4 showed high expression of the differentiation markers MUC2 and ALPI in ATcM as well as in control conditions. In stem cell-enhancing growth medium, however, ATcM from both unaffected mesenteric adipose tissue and creeping fat induced a profound alteration of organoid morphology, perfectly reflecting so-called spheroids. Additionally, this ATcM-treated organoids were larger than the control, which corresponded with increased proliferation as measured by resazurin assay. This stem-like phenotype was further confirmed by a reduced expression of the differentiation markers MUC2 and ALPI in ATcM-treated organoids compared to the control.

Conclusion

Overall, our data suggest that factors from human mesenteric adipose tissue have profound effects on intestinal epithelial stem cells, while differentiated cells appear less affected. In the stem cell niche, they promote stem cell proliferation and inhibit differentiation. In Crohn’s disease, factors released from creeping fat could thus play a significant role in the pathophysiology, e.g. by enhancing epithelial regeneration via a promotion of stem cell proliferation.
P059 Diet controls segmented filamentous bacteria in driving Crohn’s disease-like inflammation in TNFdeltaARE mice
Authors: Metwaly A , Calasan J , Waldschmitt N , Khaloian S , Häcker D , Ahmed M , Butto L F , Hammoudi N , Le Bourhis L , Mayorgas A , Siebert K , Basic M , Zeissig S , Schwerd T , Allez M , Panes J , Salas A , Bleich A , Cominelli F , Haller D
Published: January 2022

Background

Crohn’s disease (CD)-like inflammation in TNFdeltaARE mice (ARE) mice is triggered by dysbiotic gut microbial communities. Similar to the therapeutic effect of exclusive enteral nutrition in CD, dietary intervention using semi-synthetic experimental diet prevents disease development in ARE mice. The aim of this study is to identify the causal microbial cues responsible for CD-like inflammation and to dissect the protective role of diet in ARE mice.

Methods

Bacterial communities in ARE and wildtype (WT) mice were profiled by 16S rRNA gene sequencing, FISH and qPCR. Germfree WT and ARE mice were colonized with segmented filamentous bacteria (SFB) from dysbiotic ARE mice. Disease activity, cytokine expression, mucosal immune cell infiltrates as well as Paneth and goblet cells phenotypes were assessed in murine ileal and colonic tissue. To assess the influence of diet on disease development, ARE mice were fed either chow, purified semi-synthetic experimental diet (SED) or fiber-rich diet. Impact of SFB on ileo-colonic inflammation was tested in additional mouse models (XIAP-/- and IL10 -/-). Mucosal biopsies from adult and pediatric IBD patients (N=407) were screened for the presence of human SFB using SFB-specific PCR primers.

Results

Under specific pathogen-free (SPF) conditions, ARE mice developed CD-like ileal inflammation which correlated strongly with increasing abundance of SFB. Mono-colonization of germfree ARE mice with SFB resulted in severe enterocolitis. Parallel to high tissue levels of TNF and IL-17, SPF-mediated inflammation was associated with neutrophil infiltration and the expansion of IFNγ expressing Th1 cells in the mucosa. Loss of Paneth and goblet cell function allowed SFB to penetrate mucus layers towards the epithelium. SED eradicated SFB and completely abolished inflammation in ARE mice. In contrast, fiber-rich diet partially restored SFB colonization and led to increased levels of inflammatory cytokines. SFB mono-colonization of GF XIAP-/- and IL10 -/- failed to induce inflammation, emphasizing host specificity to the mucosa-adherent SFB. Presence of SFB was confirmed in 12 ileal and 11 colonic mucosal biopsies from IBD patients with active or inactive disease.

Conclusion

We identified a novel pathogenic role of SFB in driving severe CD-like ileo-colonic inflammation characterized by loss of Paneth and goblet cell functions in ARE mice. Detection of SFB in mucosal biopsies of patients with IBD opens new perspectives about therapeutic strategies targeting SFB-mediated processes. Purified diet antagonized SFB colonization and prevented disease development in ARE mice in contrast to fiber-rich diet, clearly demonstrating the important role of diet in modulating a novel IBD-relevant pathobiont.
P065 Determination of the cytokine pattern of human colon organoids derived from Inflammatory Bowel Disease patients
Authors: Molnár T , Jójárt B , Resál T , Szántó K , Kata D , Földesi I , Molnár T , Maléth J , Farkas K
Published: January 2022

Background

Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract which is associated with imbalanced pro- and anti-inflammatory cytokines. Anti-TNF-α therapy is widely used, but 10–30% of the patients are primarily not responding to the treatment or lose response over time. In vitro organoid cultures (OCs) generated from tissue specific adult stem cells can mimic cellular diversity and function of the organ of origin and might be used as a predictive tool for patient specific therapeutic response. However, the reductionist nature of the OCs and lack of knowledge about the proper representation of the disease features currently limit the utilization of the OCs as ex vivo IBD models.

Methods

Therefore our aims were to determine and compare cytokine profiles of colonic biopsies collected from IBD patients and OCs generated from the same biopsies, and to follow the changes of cytokine expression over time.

In this study samples from 20 IBD and 8 non-IBD patients were used. Biopsies were taken during colonoscopy from inflamed part of the colon of IBD patients. Crypts were isolated from biopsy samples to establish colon OCs. Total protein and RNA were isolated from biopsies and OCs. Cytokine Array was used to determine cytokine patterns in our samples. Gene expressions of control and IBD OCs after first passage was compared by qPCR. TNF-α concentrations and cellular expression was determined by EILSA and immunostainings, respectively in control and IBD OCs. To determine therapeutic response OCs were treated with anti-TNF-α therapy.

Results

Cytokine patterns of colon biopsies and OCs were remarkably similar until first passage. The major pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were detected both in biopsies and OCs. After second passage the cytokine expression decreased or disappeared in OCs. TNF-α was also measurable in the OCs, however a similar decrease was observed, which was also confirmed by immunostaining. Gene expression analysis in IBD OCs showed an increased TNF-α, IL-1β and IL-6 levels compared to healthy control OCs after first passage. ELISA also showed higher TNF-α concentration in organoids from inflamed origin compared to controls. After anti-TNF-α treatment we detected a decrease in the IL-6 gene expression in treated OCs.

Conclusion

Our results suggest that colon OCs maintain the cytokine expression ex vivo until the 2nd passage and show inflammatory characteristics. Moreover, in vitro treatment induces changes in the cytokine expression. Based on these results the utility of patient-derived organoids to predict the therapeutic response can be investigated.
P066 Lower serum concentration of sulfated bile acids is associated with Primary Sclerosing Cholangitis Inflammatory Bowel Disease comorbidity and advanced liver fibrosis
Authors: Leibovitzh H , Nayeri S , Borowski K , Hernandez-Rocha C , Lee S H , Turpin W , Stempak J , Sandhu I , Milgrom R , Smith M I , Croitoru K , Hirschfield G M , Gulamhusein A , Silverberg M S
Published: January 2022

Background

Primary sclerosing cholangitis associated Inflammatory bowel disease (PSC-IBD) carries significant morbidity risk. Alterations in bile acid profiles has been shown to modulate chronic inflammation in IBD, but data in PSC-IBD comorbidity is scarce. We aimed to assess associations of serum and stool bile acids (BA) with PSC-IBD phenotype versus ulcerative colitis (UC) and the degree of liver fibrosis in PSC-IBD patients

Methods

In this cross-sectional study, bile acids were profiled in 52 PSC-IBD and 50 UC subjects. Quantitation of bile acids was performed by reversed phase ultrahigh performance liquid chromatography/multiple reaction monitoring mass spectrometry with negative ion detection (TMIC, Alberta, CA). After data filtering and normalization, 71 serum and 76 stool BA were available for analysis. Logistic regression was used to assess associations between BA levels and disease phenotype (PSC-IBD versus UC). Among the PSC-IBD group, linear regression was used to assess associations of fibrosis degree (severe (F4) versus no/mild (F0-3) fibrosis, measured by FibroScan®) with BA profile. Age, sex and ursodeoxycholic acid treatment were included as co-variates in all models and alkaline phosphatase (ALP) was added as co-variate for the liver fibrosis analysis. False discovery rate<0.05 was considered significant

Results

PSC-IBD and UC group had similar age and sex distribution. In the multivariable logistic regression, 26 serum BA were significantly associated with PSC-IBD (Figure 1). Decreased concentrations of taurine/glycine-primary BA [odds ratio(OR)=0.97, 95% confidence interval (CI) 0.95–0.99], taurine/glycine-secondary BA [OR=0.98, 95%CI 0.97–0.99] and sulfated-BA [OR=0.96, 95%CI 0.95–0.98], and increased concentrations of conjugated-BA [OR=1.02, 95%CI 1.01–1.03] and glucuronidated-BA [OR=1.06, 95%CI 1.02–1.1] were associated with PSC-IBD phenotype (q<0.05). Decreased sulfated-BA concentrations remained associated with PSC-IBD [OR=0.96, 95%CI 0.94–0.99, q=0.06] even among PSC-IBD subjects with normal ALP<2XULN (n=67). In contrast, stool BA analysis did not show significant associations with PSC-IBD versus UC. Additionally, in the PSC-IBD group, F4 fibrosis degree compared to F0-3 was associated with lower serum concentrations of taurine/glycine- and sulfated-BA, and higher serum concentrations of conjugated-BA (q<0.05)

Conclusion

Decreased concentration of sulfated-BA was associated with PSC-IBD phenotype with and without cholestasis compared to UC group. Lower sulfated-BA concentration was also associated with severe degree of liver fibrosis among PSC-IBD subjects. This inverse relationship between sulfated-BA levels and PSC-IBD and the degree of fibrosis should be further explored in future mechanistic studies
P076 Patients with Inflammatory Bowel Disease show IgG immune responses towards disease-associated small intestinal bacteria
Authors: Bourgonje A R , Roo-Brand G , Lisotto P , Sadaghian Sadabad M , Reitsema R D , de Goffau M C , Faber K N , Dijkstra G , Harmsen H J M
Published: January 2022

Background

Inflammatory bowel disease (IBD) is characterized by a disturbed gut microbiota composition. Patients with IBD have elevated levels of mucosal and serum levels of IgG-antibodies directed against bacterial antigens, including flagellins. In this study, we aimed to determine to which faecal bacteria the humoral immune response is directed to in patients with IBD.

Methods

Faecal and serum samples were collected from patients with IBD (n=55) and age- and sex-matched healthy controls (n=55). Faecal samples were incubated with autologous serum and IgG-coated fractions were isolated by magnetic-activated cell sorting (MACS) and the coating efficiency was assessed by flow cytometry. Bacterial composition of both untreated and IgG-sorted fecal samples was determined by 16S rRNA-gene Illumina sequencing.

Results

Serum IgG responses were primarily directed to typical small intestinal bacterial genera, including Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Veillonella and Enterobacteriaceae, as well as against specific Lachnospiraceae bacteria, including Coprococcus and Dorea (all P<0.001) (Figures), and to the species Ruminococcus gnavus (P<0.05). In contrast, serological IgG responses against typical commensal, anaerobic and colonic microbial species were rather low, e.g. to the Lachnospiraceae members Roseburia and Blautia, to Faecalibacterium as well as to Bacteroides. IgG-sorted faecal samples were characterized by significantly lower microbial diversity. Patients with IBD showed more IgG-coating of Streptococcus, Lactobacillus and Lactococcus bacteria compared with healthy controls (all P<0.05). No differences in IgG-coated bacterial fractions were observed between CD and UC, between active or non-active disease, nor between different disease locations in CD.

Conclusion

The IgG immune response is specifically targeted at typical small intestinal bacterial genera, whereas responses against commensal, rather colonic-type microbiota are lower in patients with IBD. These findings may be indicative of a strong immunological exposure to small intestinal bacteria in concordance with relative immune tolerance against commensal bacteria.
P092 Humoral and T cell responses to COVID-19 vaccination in IBD
Authors: Zhang E , Bond K , Nguyen O , Allen L , Kedzierski L , Kedzierka K , Christensen B
Published: January 2022

Background

The characteristics of SARS-CoV-2 vaccine-induced immunity in inflammatory bowel disease (IBD) patients on immune modifying agents has not been clearly defined due to their exclusion in vaccine trials. Emerging results suggest infliximab impairs antibody response compared to vedolizumab. However there has not been direct comparison to controls. We evaluated this with both humoral and T cell response in IBD patients.

Methods

Antibody and T cell response were analysed in IBD patients who received BNT162b2 (Pfizer–BioNTech) or ChAdOx1 nCoV-19 (Oxford–AstraZeneca) vaccination from a single Australian centre. The control group were healthcare workers (HCW) without IBD. Blood samples were taken at 4 time points: at baseline V0 (before vaccination); V1 (7–14 days after vaccine 1); V2 (7–14 days after vaccine 2); V3 (21–42 days after vaccine 2). Antibodies to the S1/2 IgG subunit and receptor-binding protein (RBD) were measured and reported here.

Results

88 (28 ulcerative colitis, 50 Crohn’s disease) IBD patients were included and compared to 53 healthy controls (Table 1). IBD patients medications included 6 5ASA (6.8%), 6 immunomodulator monotherapy (6.8%), 14 anti-TNF monotherapy (15.9%), 32 anti-TNF combination therapy with immunomodulator (36%), 16 IL12/23 (18%) and 13 vedolizumab (14%). Pre-vaccine baseline sera showed absence of anti-RBD antibodies in all participants. 84 patients (87%) received BNT162b2 and 4 (4.5%) received ChAdOx1 nCoV-19 vaccines. Geometric mean [SD] anti-S1/2 antibody concentrations at 4 weeks after second vaccination (V3) were significantly lower in IBD TNF treated patients (162.6[1.7]) compared to IBD non TNF treated patients (325.2[1.3]), and healthy controls (325.2[1.3]), p<0.0001 (Figure 1). There was no difference between non-TNF treated patients including those on vedolizumab or IL12/23 compared to controls. Similarly there was a significant difference between anti-RBD IgG titres between TNF and non-TNF IBD patients at V3 but not when compared to controls (Figure 2). There was no difference in RBD IgG and anti-S1/2 antibodies between anti-TNF monotherapy and combination anti-TNF with immunomodulator. All IBD and healthy controls seroconverted at V3 (Figure 3).

Conclusion

TNF agents influence SARS-CoV-2 vaccine-induced antibody response in IBD patients, with lower anti-S1/2 IgG concentrations compared to non-TNF IBD patients and healthy controls. However, there was no difference in RBD IgG titres between controls and IBD patients overall. It is unclear whether these subtle differences in antibody response in IBD patients on TNF agents is biologically meaningful, as all groups seroconverted after second dose vaccination. Neutralising antibody and T cell data (CD4+/CD8+) from this study to come.
P094 Transcriptomic and small RNA sequencing profile in ileal resections from complicated Crohn′s Disease patients
Authors: Seco-Cervera M , Bauset C , Santana-Plata C , Civera-Barrachina M , Ortiz-Masià D , Calatayud S , Barrachina M D
Published: January 2022

Background

Crohn′s disease is a chronic inflammatory disorder of gastrointestinal tract that is classified into three different behaviours: the inflammatory (B1), the stenotic (B2) or the penetrating (B3). We pretend to identify differences in transcriptomic and non-long coding RNA expression profiles associated to damaged and no-damaged surgical ileal resections from complicated CD patients.

Methods

We conducted both RNA and small RNA sequencing profiling on ileal surgical resections from CD patients with structuring (n=10) or penetrating (n=10) behaviour; from each patient we obtained a sample from affected tissue (B2A and B3A, respectively) and the paired non-affected ileum (B2C and B3C, respectively). Ten ileal resections (control samples) were obtained from non-affected ileum of patients with right colorectal cancer (IL). Enrichment scores of the GO terms and KEGG pathways were used to functional analysis investigated in this study. Gene expression and miRNA profiles were validated by RT-qPCR.

Results

Our comparative bioinformatic analysis of RNA sequencing showed that the comparation of IL and the non-affected ileum from CD patients (B2C+B3C) revealed that only 11 genes were significantly altered and any miRNA with significative expression. The comparative analysis between B2A+B3A vs B2C+B3C showed: a) 2562 genes differentially regulated and the GO enrichment analysis determined that inflammation, cell activation, and regulation of the extracellular matrix (EM) were the main biological processes altered (Figure 1a). KEGG analysis showed that cytokine-receptor interaction and MAPK and Ras signalling were the most affected pathways (Figure 1b). Among the top ten up-regulated genes we found several immunoglobulins (such as IGHG4, IGHG3, and IGHG1), proteins of the extracellular matrix EM (such as EPYC, COMP, and CHI3L1), and transcription factors (such as PRRX1). When the analysis was performed by CD behaviour, results revealed that most of these genes were significantly increased in both, B2 and B3 CD; b) 103 miRNAs with differential expression, 56 were up-regulated and 47 were down-regulated. Negative and significant correlations were detected between the RNA expression of ECM components and some miRNA expression.

Figure 1. Bar plot of enrichment analysis with differential expressed genes between B2A+B3A vs B2C+B3C. A) Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) of Gene Ontology (GO) enrichment analysis. B) Enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

Conclusion

The correlation detected between extracellular matrix components and some miRNA suggest an epigenetic regulation in the affected ileum of complicated CD patients.
P099 Serum proteomic analysis reveals novel biomarkers associated with disease activity in pediatric patients with Crohn’s disease
Authors: Werner L , Weiss B , Matar M , Shamir R , Shouval D
Published: January 2022

Background

Although Crohn’s disease (CD) is associated with a marked hyper-inflammatory response, identifying serum biomarkers associated with disease activity and outcomes is considered challenging, given lack of sensitivity of different assays. Our aim was to identify novel markers using Olink, a novel technology based on proximity extension assay, enabling detection of minute amounts of proteins.

Methods

Serum samples were obtained from pediatric patients with CD at the time of diagnosis and following induction therapy, and from control subjects, consisting of pediatric patients with normal endoscopic procedures, without past or present history of an immune-mediated disorder (e.g. celiac, diabetes). Serums were subjected to Olink (two panels used, consisting of 184 proteins), and analysis was performed on a Normalized Protein eXpression file by supervised, multivariate, principal component analysis and verification by univariate ANOVA with Benjamini-Hochberg and post-hoc Tukey analysis.

Results

Eighty-eight serum samples were collected: 30 from control subjects and 58 from 32 patients with CD (24 of them pre- and post-induction therapy). The median age of patients in the control and CD groups was 13.9 years (IQR 11.1–16.6) and 14.6 years (IQR 12.2–16.9), respectively (P=0.32). Twenty-four patients were treated with anti-TNFa agents and 8 with EEN. The median pediatric Crohn’s Disease Activity Index (PCDAI) decreased from 35 (22.5–42.5) to 5 (0–12.5, P<0.001) following induction therapy. We identified 72 proteins that significantly differed between patients and controls, and many of them were associated with CRP or ESR levels. String analysis identified several important nodes linking different proteins, including MMPs, MPO, EGFR and TNFRSF1A. Many proteins, such as resistin and different MMPs, showed a strong positive correlation with disease activity, based on PCDAI, while others, such as EGFR, showed a negative correlation. Several novel markers were identified to be highly expressed in the serum of patients with CD, including LRIG1, an intestinal stem cell marker, that was highly correlated with disease activity among the patients.

Conclusion

We identified novel serum markers that are associated with disease activity in pediatric patients with CD. These findings should be validated in future studies, and be complemented with expression and functional studies to define their role in mediating intestinal immune responses.
P105 Treatment of the lower gastrointestinal tract Klebsiella infections by recombinant bacteriocin KvarIa
Authors: Karaliute I , Ramonaite R , Kupcinskas J , Misiunas A , Denkovskiene E , Gleba Y , Razanskiene A , Skieceviciene J
Published: January 2022

Background

Hospital-acquired infections (HAI) are one of the leading worldwide health care issues. The most prevalent HAI are the Klebsiella species. K. quasipneumoniae strains tend to colonize the gastrointestinal tract (GI) and show high rates of resistance against antibiotics. Orally delivered recombinant bacteriocins, like klebicins, could be employed as oral antimicrobials to eradicate multidrug-resistant Klebsiella from the intestinal tract prior to/during hospitalization.

Methods

This study aimed to investigate the antimicrobial efficacy of orally delivered Eudragit - coated klebicin (KvarIa) in a murine lower gastrointestinal tract model of K. quasipneumoniae infection. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy, and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated KvarIa. Faecal and tissue samples were used for Klebsiella haemolysin gene (khe) analysis using RT-PCR.

Results

The gastrointestinal K. quasipneumoniae colonization was achieved only in the antibiotic-treated mice groups ((i) penicillin, streptomycin or (ii) penicillin, streptomycin, and metronidazole) followed by the introduction of K. quasipneumoniae. Analysis of the antimicrobial efficacy of Kvarla showed that coated-KvarIa significantly reduced colonization of K. quasipneumoniae in both mice groups compared to the control group. The amounts of K. quasipneumoniae changed from 6.3 x 107 CFU/50mg on the 18th day to 3.9 x 105 CFU/50mg on the 22nd day (p = 0.01) in the Eudragit S100-coated KvarIa 100 µg group and from 4.0 x 107 CFU/50mg on the 18th day to 1.6 x 105 CFU/50mg on 22nd day (p = 0.009) in the Eudragit S100-coated KvarIa 1000 µg group.

Conclusion

Our study demonstrates that colonization of mice intestinal tract by K. quasipneumoniae can be achieved after eradication of resident gut microbiota with antibiotics. Furthermore, using this murine model, we showed that orally delivered encapsulated klebicins substantially reduce K. quasipneumoniae colonization in the lower gastrointestinal tract.
P110 Risk Factors of Clinical Relapses in Pediatric Luminal Crohn’s Disease, a Retrospective Cohort Study
Authors: Sassine S , Djani L , Cambron-Asselin C , Savoie-Robichaud M , Lin Y F , Qaddouri M , Fadela-Zekhnine S , Grzywacz K , Groleau V , Dirks M , Drouin É , Halac U , Marchand V , Girard C , Courbette O , Patey N , Dal Soglio D , Deslandres C , Jantchou P
Published: January 2022

Background

Few risk factors are associated with the risk of relapses of Crohn’s disease in children. The aims of this retrospective cohort study were to describe the rate of relapses in children with Crohn’s disease, its evolution over the past decade and to determine risk factors associated with relapse.

Methods

Patients under 18 years old and diagnosed between 2009 and 2019 were included. Patients clinical, endoscopic, histological, and laboratory characteristics, as well as their treatments, where collected from their medical records and the prospective CHU Sainte-Justine inflammatory bowel disease registry. Survival analyses and Cox regression models were used to assess the impact of those risk factors on relapse.

Results

639 patients were included. There was a decrease in the clinical relapse rate over the past decade: 70.9% of patients diagnosed between 2009 and 2014 experienced a relapse compared to 49.1% of patients diagnosed between 2015 and 2019 (p<0.0001). The following variables were associated with clinical relapse: female sex (adjusted hazard ratio (aHR)= 1.51, p=0.0009), high PCDAI (aHR= 1.02, p=0.04) and SES-CD (aHR= 1.03, p=0.03) scores at diagnosis, upper digestive tract involvement (aHR= 1.59, p=0.0003), exposure to oral 5-ASA (aHR= 1.91, p=0.0003), use of immunomodulatory agents compared to TNF-alpha inhibitors (methotrexate aHR= 1.91, p=0.0006; thiopurines aHR= 2.06, p<0.0001), presence of granulomas (aHR= 1.27, p=0.04) and increased eosinophils on intestinal biopsies (aHR= 1.34, p=0.02), high levels of C-reactive protein (aHR= 1.01, p<0.0001) and fecal calprotectin (aHR=1.09, p<0.0001) during clinical remission and low serum infliximab levels during maintenance (aHR for mean serum infliximab level under 7ug/mL = 2.48, p=0.005).

Figure 1: Kaplan-Meier curve representing patients time to relapse according to the first maintenance treatment administered.

Conclusion

Relapse risk was significantly associated with baseline clinical, endoscopic, histological and laboratory data and treatment strategies. These results could help better select treatment options for pediatric Crohn’s disease at induction and maintenance.

Figure 2: Kaplan-Meier curve representing patients time to relapse according to the mean infliximab level in post-induction.