bluuebunny/crossref_metadata_2025 huggingface dataset:
P006 3D Multicellular intestine-on-a-chip model for disease modelling and drug discovery
Authors: Lo Sasso G , Gijzen L , Marescotti D , Naumovska E , Raineri E , Nicolas A , Lanz H , Guerrera D , van Vught R , Joore J , Vulto P , Peitsch M C , Hoeng J , Kurek D
Published: January 2020

Background

One of the major functions of the human intestine is to provide a protective epithelial barrier between the body and digestive environment. Additionally, the interplay of commensal microbes of the gut microbiome with the gut tissue and host immune system significantly contributes to intestinal homeostasis. Crohn’s disease and ulcerative colitis, collectively referred to as inflammatory bowel diseases, are both associated with increased permeability of the epithelial barrier and dysregulated immune response. Great efforts have been made to develop both in vitro and in vivo models of the human intestine in order to understand the development and underlying pathogenesis of IBD. These efforts have provided valuable insights into multiple aspects of the disease. However, none of these models has been able to capture the complexity and multifactorial nature of IBD. Animal models generally fail to accurately predict the efficacy and toxicity of novel compounds in human tissues, while in vitro human intestinal models developed on porous membranes within Transwell inserts fail to accurately recapitulate and mimic key aspects of the in vivo situation.

Methods

Here, we present the development and characterisation of a 3D multicellular perfused intestine-on-a-chip model in a microfluidic platform, the OrganoPlate®, and its application for investigating intestinal inflammation. The model described here comprises a coculture of Caco-2 and mucus-secreting HT29-MTX cells in the top compartment of the chip and a coculture of immune-competent cells THP-1 and MUTZ-3 in the bottom compartment, lining a collagen-I ECM in the middle.

Results

We show that the Caco-2 and HT29-MTX coculture form confluent and polarised tubular structures against the collagen-I ECM in the OrganoPlate®, with a stable barrier function over time as well as the capability to secrete mucus. By exposing the cultures to TNFα and/or IL-1β, we were able to induce an inflammatory state, characterised by cytokine release (IL-8) and a decrease in trans-epithelial electrical resistance. Finally, we proved the applicability of the model in screening anti-inflammatory compounds by its reversibility. Using a well-known anti-inflammatory drug, TPCA-1, we were able to prevent cytokine-induced inflammation. This result was evident from the decreased secretion of IL-8 and retention of barrier function in treated cultures, similar to that observed in untreated cultures.

Conclusion

Overall, this complex 3D multicellular perfused intestine-on-a-chip model provides the versatile modularity of mimicking key features of intestinal inflammation and can, therefore, further support drug screening efforts and provide a platform for personalised medicine.
P007 GC1qR driven oxidative phosphorylation is essential for intestinal goblet cell differentiation
Authors: SÜNDERHAUF A , Hicken M , Skibbe K , Schlichting H , Hirose M , Perner S , Ibrahim S , Derer S , Sina C
Published: January 2020

Background

Induction of goblet cell differentiation during inflammation has been shown to be impaired in ulcerative colitis (UC) but not Crohn’s disease (CD), possibly explaining the intestinal goblet cell and mucus reduction observed in active UC. A metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS) is necessary for terminal differentiation of intestinal stem cells towards goblet cells. Interestingly, intestinal energy deficiency in general and reduced level of OXPHOS in specific have been attributed to UC pathogenesis more than 30 years ago. The c1q binding protein (C1QBP; gC1qR) is indispensable for the maintenance of OXPHOS. Nevertheless, experimental evidence linking mitochondrial dysfunction with goblet cell depletion and C1QBP expression are still missing.

Methods

Goblet cell differentiation was studied in human biopsies from UC patients in remission, in mucus-producing HT29MTX cells and in a conplastic mouse strain with diminished mitochondrial OXPHOS activity. Furthermore, mice were fed an experimental diet to shift cellular energy production from glycolysis to OXPHOS and mucosal cell differentiation was compared with mice on an isocaloric control diet.

Results

In vitro, siRNA experiments in HT29MTX cells showed that butyrate-induced expression of goblet cell differentiation factor KLF4 is highly dependent on gC1qR expression. Interestingly, the latter was significantly reduced in human ileal and colonic sections of UC patients in remission compared with HN. OXPHOS-deficient conplastic B6-mt FVB mice further confirmed these findings by depicting diminished klf4 expression, lowered goblet cell numbers, a thinned intestinal mucus layer and signs of intestinal inflammation. Finally, via nutritional intervention in C57BL/6 mice we were able to increase gC1qR level and to induce goblet cell differentiation via hath1 and klf4 compared with controls.

Conclusion

Taken together, we here describe a new pathway linking low intestinal expression of OXPHOS-regulating gC1qR to impaired goblet cell differentiation, mucus reduction and mucosal inflammation, which could be possibly reversed by nutritional intervention.
P008 Whole-genome RNA sequencing in collagenous colitis: a subgroup analysis
Authors: Escudero-Hernández C , van Beelen Granlund A , Bruland T , Sandvik A K , Koch S , Østvik A E , Münch A
Published: January 2020

Background

Collagenous colitis (CC) is a common inflammatory bowel disease that exhibits chronic watery diarrhoea. Treatment with the glucocorticoid budesonide is effective to induce and maintain clinical remission in most cases; however, few patients do not respond to treatment. To distinguish between different CC subgroups on a molecular level, we performed genome-wide RNA sequencing (RNAseq) analysis on mucosal samples.

Methods

We collected colonic biopsies from healthy controls (Hc) and CC patients with active disease. Additionally, we obtained matched samples from budesonide-responsive patients (clinical remission) under treatment with budesonide (9 mg/day, 8 weeks), and biopsies from budesonide-refractory patients (n = 9 patients/group). Ulcerative colitis (UC) samples were included as a separate control. Total RNA was isolated for RNAseq. Whole-genome expression data were analysed by principal component analysis (PCA) and differential gene expression (DGE) using R software. The Benjamini–Hochberg FDR method adjusted p values, considering <0.05 as statistically significant. Gene-set enrichment analysis (GSEA) was performed using GSEA software and visualised in Cytoscape. Results were validated in tissue samples from the same patients by immunohistochemistry (IHC).

Results

PCA of all samples identified three principal components which separated sample groups into distinct clusters of gene expression, explaining 31% of the transcriptional variation. PCA clearly demarcated UC samples from Hc and CC. Mucosal samples from budesonide-refractory patients exhibited a discrete RNA expression profile that was distinct from all other groups. Moreover, PCA of budesonide-responsive persons also separated active from treated CC. Subsequent DGE analysis revealed differential expression of 395 genes in patients with active CC compared with Hc, and that these genes were mainly involved in immune response, cell cycle and apoptosis, according to GSEA. Using IHC, we confirmed the impaired proliferation and immune response on the protein level. In samples from budesonide-treated patients, 75 genes were differentially regulated compared with controls. A paired comparison of matched samples from CC patients before and after induction of remission showed an expression change of 337 genes, mainly involved in DNA recognition.

Conclusion

CC is a transcriptionally homogeneous disease that can be characterised by differential gene expression. Unique gene expression profiles describe patient sub-populations, which could define budesonide-refractory CC as a distinct disease entity. Further study of the transcriptional landscape of CC may reveal pathogenic mechanisms and therapeutic targets for this common, debilitating inflammatory bowel disease.
P013 The JAK-3 and TYK-2 / STAT pathways are activated in moderate to severe ulcerative colitis
Authors: Loza M , Brea J M , Calviño-Suarez C , Baston-Rey I , Ferreiro-Iglesias R , Dominguez-Munoz J E , Barreiro-de Acosta M
Published: January 2020

Background

Ulcerative colitis (UC) is a chronic, progressive and disabling disease with a complex pathology of unknown aetiology influenced by genetic, environmental and microbiota factors that lead to an immunological and inflammatory response in the colon. Janus Activated Kinase (JAK) family plays a key role in modulating the adaptive and innate inflammatory response. The JAK/STAT pathway involvement in UC has been demonstrated in both animal models and human studies. Thus, overexpressed JAK-3 has been detected in the intestine of patients with UC, suggesting a key role in their pathophysiology and the inhibition of TYK-2 in animal models resulted in an improvement of the disease, which would explain its implication in the inflammatory process. We hypothesise here that there could be an activation of JAK-3 and TYK-2 signalling pathways in UC patients. Thus, we aimed to detect the activation of both signalling pathways by means of western-blot studies in UC patient samples

Methods

A prospective, observational single-centre study was designed. Inclusion criteria were adult patients with endoscopic active UC (more than Mayo-0) confirmed in a programmed colonoscopy. All patients signed informed consent. Samples were obtained from overstock of routine biopsies in the more severe segment affected of the large bowel. Tissues were homogenised and processed in order to obtain cell lysates by employing RIPA buffer and ultrasounds. The degree of activation of the JAK-3 and TYK-2 pathways was measured by detecting the phosphorylation of both targets as well as of STAT1, STAT3, STAT4, STAT5 and STAT6 through western blot by employing specific antibodies for total and phosphorylated proteins.

Results

19 UC patients were consecutively included. Mean age was 46 years old. 53% were female, 47% were extensive colitis (E3) and 53% left-side colitis (E2). Regarding endoscopic activity, 26% had Mayo-1, 53% Mayo-2, and 21% Mayo-3. Immunoreactive bands for both phosphorylated JAK-3 and TYK-2 were detected in the biopsies from UC patients, evidencing that colonic inflammation leads to an activation of both targets. The study of STATs phosphorylation showed immunoreactive bands for phosphorylated forms of STAT1, STAT3, STAT4, STAT5 and STAT6 confirming the activation of both signalling-pathways in these patients (Figure 1).

Conclusion

The developed translational workflows involving basic/clinical research confirm the activation of both JAK-3 and TYK-2-dependent signalling pathways in UC patients, validating both kinases as targets for treating UC. The developed methodology allows studying the target engagement for future JAK-3/ TYK-2 inhibitors employed in clinical trials.
P016 Efficiency of evaluating the expression of Toll-like receptors 2, 4, 6 and proteins proteomic profiling as integral indicators for predicting the risk of early relapse of ulcerative colitis
Authors: Tarasova G , Volkov A , Iakovlev A , Ghazudin T , Shcherbakova I
Published: January 2020

Background

Success was made in the study of TLR in congenital and adaptive immunity, which determined a new look at immune processes at ulcerative colitis (UC). Modern achievements of proteomic methods of analysis research allow to define molecular characteristics of the inflammation in colon mucosa of patients with UC.

Methods

The study included 86 patients with UC, an average age of 39.0 ± 1.4 years. Groups: 1–15 (17.4%) patients with distal form of UC, 2–42 (48%) left-sided form, 3–29 (33.7%) patients with total UC. The expression of TLR on peripheral blood monocytes was determined in the immunofluorescence test. Two-colour analysis was performed on a flowthrough laser cytofluorimeter (Cytomics FC500, Beckman Coulte). The percentage of monocytes (CD14 + cells) carrying TLR2, TLR4, and TLR6 on their surface was assessed. The separation of proteins of colon mucosa was based on technologies of IEF, SDS–PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, Bruker, USA). The getting of mass spectrogram was determined by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, Bruker, USA). Statistical analysis was performed using the software Statistica 10.0 (Statsoft).

Results

The direct average relationship was established between the number of monocytes expressing CD14 + CD282 +, CD14 + CD284 +, CD14 + CD286 + and the area of inflammation (r = 0.49, r = 0.55, r = 0.42, p < 0.05). The nonlinear regression equation was used. Calculation example: the risk of recurrence UC = exp (−26.1 + (0.4) × TLR 2)/(1 + exp (−26.1 + (0.4) × TLR 2)), χ2 = 130, 59, p < 0.0001. Thus, when the number of monocytes expressing TLR2 is not more than 60%, the risk of recurrence of the UC is not more than 11%, with values above 70%, the probability of recurrence exceeds 80%. We identified potentially new molecular markers of the early relapse of ulcerative colitis: SMAD2 activates the transcription of TFG1β and leads to development of fibrosis in colon submucosa in patients with UC; significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signalling pathways that promotes the increase of the synthesis of IL-2, 6, 8, 12, TNFα, the activity of immune and inflammation processes in colon mucosa; the reduction of expression of β-defensin-1 in cells of colon mucosa is accompanied with increased expression of CCR6 that promotes the formation of inflammatory infiltrates in colonic submucosa in UC.

Conclusion

Expression of TLR 2, 4, 6 in blood monocytes (the risk of recurrence UC ratio) and new protein molecular markers of colon mucosa (SMAD2, PPARγ, and apoС-III) can be used as a tool for prediction of early relapse UC.
P019 Colonic mucosal kinase activity, cytokine and chemokine profiles in inflammatory bowel disease
Authors: Brand E , Roosenboom B , Malvar Fernandez B , Lutter L , van Koolwijk E , van Lochem E G , Horjus Talabur Horje C S , Reedquist K A , van Wijk F , Oldenburg B
Published: January 2020

Background

With the approval of tofacitinib, an oral Janus Kinase (JAK) inhibitor, modulation of kinase activity has been added to the therapeutic armamentarium of inflammatory bowel disease (IBD). Despite its established efficacy, at least a third of patients will not respond to this or other therapeutic options such as anti-tumour necrosis factor (TNF), anti-interleukin (IL)23/IL12 compounds or vedolizumab. A better understanding of the inflammatory profile could aid in tailoring drugs to individual patients. We therefore explored mucosal cytokine, chemokine and kinase activity profiles in IBD.

Methods

Colonic mucosal biopsies were collected from (1) patients with Crohn’s disease (CD, N = 8), (2) patients with ulcerative colitis (UC, N = 8) and (3) healthy controls (N = 4). IBD samples were collected both from inflamed and non-inflamed tissue from the same patients. All IBD patients were biological-naïve and had not used corticosteroids in the past 3 months. Biopsies were snap frozen for later kinase activity determination or directly used in a 24-h explant culture. Whole biopsy kinase activity (tyrosine, serine and threonine kinases) was assessed using the Pamgene platform. A 64-analyte panel was examined in the supernatant of the cultured biopsies employing a multiplex assay (Luminex).

Results

Whole-biopsy kinase activity differed between inflamed and non-inflamed mucosa of IBD patients, with more overall tyrosine kinase activity in inflamed mucosa in UC, and serine/threonine kinase activity in inflamed mucosa in CD as compared with non-inflamed mucosa (Figure 1). The kinase activity profile of non-inflamed mucosa of CD and UC patients was similarly different from mucosa of healthy control participants (Figure 2). The cytokine and chemokine profile of inflamed biopsies differed from non-inflamed IBD biopsies and healthy control biopsies, with higher levels of S100A8, TNFα, IL-6, oncostatin M (OSM) and triggering receptor expressed on myeloid cells-1 (TREM-1), amongst others (Figure 3).

Conclusion

In IBD, inflammation in the mucosa can be characterised both by explant-culture and kinase activity assessment. The difference in kinase activity between non-inflamed IBD mucosa and healthy control mucosa suggests the presence of sub-clinical alterations in cell signalling. The observed differences in the kinase, cytokine and chemokine profiles underscore the importance of this approach in the elucidation of the pathophysiology in IBD.
P022 Impact of increasing disease symptom concordance between patients with inflammatory bowel disease and their physicians: insights from US, France, Germany, Italy, Spain and UK (5EU)
Authors: Kershaw J , Wild R , Lukanova R , Bruce Wirta S , Sikirica M
Published: January 2020

Background

Accurate disease activity assessment builds on dialogue between patients with CD and UC and their doctors and is key for management of IBD.

Objective

Assess the level of patient and physician symptom concordance on disease, health-related quality of life (HrQol), work productivity and patient satisfaction using the Adelphi Disease-Specific Programme.

Methods

Cross-sectional study among CD and UC patients and their gastroenterologists in US and 5EU. Data were collected in two waves, from November 2014–March 2015, and September 2017–January 2018, and then merged. Physicians completed patient record forms (PRFs) for the next seven UC and eight CD patients; same patients completed patient self-completion (PSC) forms describing how UC/CD affected them. PRFs collected diagnosis data, disease history, symptoms, treatment and management history. PSCs collected similar data, including patient reported outcome measures to assess the humanistic burden. Patients were grouped based on degree of alignment with physicians in symptom reporting (full agreement, discordance on 1–4 symptoms, discordance on 5+ symptoms out of 22 symptoms). Comparisons between groups were made using Chi-squared and Kruskal–Wallis for continuous and categorical variables.

Results

A total of 2524 CD and 2153 UC patients were included in the analysis (mean age CD 38.5/UC 40.3, CD 50.2% male/UC 52.0%, mean time since diagnosis CD 5.6 years/UC 5.1 years). There was overall reasonable agreement in symptom reporting between patients and their physicians; however, amongst 17% of UC and CD patients and physicians, there was discordance on 5+ symptoms. Higher symptom concordance significantly associated with better patient outcomes: higher remission rates, productivity, HrQoL scores and satisfaction (Table 1).

Conclusion

We observed moderate discordance between patient and physician reporting of current UC and CD symptoms. While even small discordance was associated with worse clinical outcomes: HrQol, productivity and satisfaction. Future studies are needed to improve understanding of the reasons behind discordance and association with worsened outcomes.
P027 Repertoire analysis of memory T-cell receptors in Japanese patients with inflammatory bowel disease
Authors: Takeru N , Kakuta Y , Izumiyama Y , Ichikawa R , Okamoto D , Moroi R , Kuroha M , Kanazawa Y , Kinouchi Y , Masamune A
Published: January 2020

Background

The T-cell receptor (TCR) repertoire was assessed in response to various antigens and was considered to be associated with the pathogenesis of inflammatory bowel disease (IBD). Thus, we performed TCR repertoire analysis to examine the pathology of IBD from changes in the TCR repertoire of memory T cells in the intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) of patients with IBD.

Methods

LPMCs in the surgical specimens and PBMCs were isolated from 12 patients with IBD [five patients with ulcerative colitis (UC) and seven patients with Crohn’s disease (CD)]. PBMCs were collected from 10 healthy individuals as controls. Comprehensive TCR sequence analyses of adaptor-ligation PCR products were performed using MiSeq.

Results

The diversity of TCR-α and -β in PBMCs was significantly lower in patients with IBD than that in controls (p = 0.00084 and 0.0013, respectively). Comparisons of TCR diversity in LPMCs and PBMCs between CD and UC showed that the diversity in LPMC was not affected by diseases, whereas that in PBMCs was significantly lower in CD than in UC (p = 0.045 and 0.049, respectively). Some TCR clones may have shown a specific increase or decrease in CD and UC and many clones were common to both LPMCs and PBMCs in the same patients.

Conclusion

The diversity of TCR clones in LPMCs and PBMCs in patients with IBD was significantly lower than that of PBMCs in controls. TCR diversity in PBMCs was particularly low in patients with CD.
P028 The anti-inflammatory effects of a poly-probiotic on the oral mucosa
Authors: Marlina E , Hirani B , Mercadante V , Shephard M , Kishida S , Sebepos-Rogers G , McMillan R , Hodgson T , Leeson R , Porter S , Barber J , Fedele S , Smith A
Published: January 2020

Background

Probiotics have previously showed evidence of being efficacious in inducing and maintaining remission in post-operative recurrent pouchitis. The potential mechanism of action of probiotics has been attributed to their ability to reduce pro-inflammatory cytokine production, both within the mucosal tissue and systemically. We present our study which characterises the anti-inflammatory effects of probiotics on the oral epithelium and in the treatment of oral lichen planus (OLP), a chronic inflammatory disease of the oral mucosa.

Methods

VSL#3 (VSL#3-ACTIVE batch 703093 Exp date 04/2019) is a highly concentrated (450 billion live bacteria per sachet) poly-probiotic food supplement that contains eight different strains of live bacteria. The mouth ordinary epithelium cell line (MOE-1a) was stimulated with VSL#3 plus or minus the pro-inflammatory bacteria E. coli. The resultant effects on cytokine production and wound healing were measured using ELISA and live cell imaging. Wound closure was calculated using ImageJ software. OLP patients (n = 80) and healthy controls (n = 44) were recruited from UCLH Eastman Dental Hospital (Ethics 17/LO/0475) and saliva and blood samples tested for CXCL10 levels using an ELISA. OLP patients with active disease (n = 30) were recruited into a double-blind placebo-controlled proof-of-concept trial investigating the potential benefit of VSL#3 in the treatment of clinical symptoms (NCT03052179). Patients consumed two sachets twice daily for 30 days with a 30-day follow-up. Clinical questionnaires, saliva and peripheral blood were collected on days 0, 30 and 60. A daily quality of life and compliance diary was used by all participants.

Results

The addition of VSL#3 to MOE1a cells stimulated with E. coli resulted in a significant reduction in pro-inflammatory cytokine secretion and an acceleration in wound healing. OLP patients were found to have an elevation in the pro-inflammatory chemokine CXCL10 both locally (saliva) and systemically (serum) compared with healthy controls. Finally, the clinical trial demonstrated that VSL#3 was tolerated and safe for patients with OLP. Although there is no statistical evidence, descriptive results suggest that VSL#3 can confer some beneficial effects on patients with active OLP. We noted a reduction in the number of sites of disease activity and an improvement in quality of life in the VSL#3 group compared with placebo. Corticosteroid usage was also reduced in the VSL#3 group.

Conclusion

VSL#3 has the ability to improve oral epithelial wound healing and reduce pro-inflammatory cytokines secretion in vitro. In OLP, the consumption of VSL#3 seems to provide some clinical benefits, but due to the study size a more substantial multi-centre trial is necessary to confirm these observations.
P029 TRIM 21 protects against ulcerative colitis and colitic cancer via smad7 inhibition
Authors: Hahm K B
Published: January 2020

Background

Proteins of the tripartite motif-containing (TRIM) superfamily are critical in a variety of biological processes in either innate immunity or eliminating invading pathogens, by which had been implicated in pathogenesis of autoimmune diseases including inflammatory bowel diseases. The typical structure of TRIM proteins contains a RING motif in the N-terminal end, followed by a B-box motif, a coiled-coil domain and a B30.2/PRYSPRY region in the C-terminal end led to the regulation of TGF-β anti-inflammatory cytokines, by which TRIM21 has been reported to regulate IBD negatively through inhibiting Th1/Th17 cell differentiation.

Methods

Since antisense oligonucleotide targeting smad7 was withdrawn from clinical trial due to insufficient efficacy, in this study, we generated TRIM21 overexpressed cell lines to study the binding of TRIM21 to smad7 as well as the regulation of consequent TGF-β receptor.

Results

TRIM21 significantly binds to smad7 as well as repressed levels of TGF-b type I/II receptor. SBE-luc and 3TP-luc assay showed significantly decreased activities under TRIM21 + TGF-β. Since TRIM21 contains ubiquitin ligase, PRYSPRY, TRIM21 with TGF-β significantly decreased TGFRII via UPL. These in vitro evidences that TRIM21 significantly repressed TGF-β after binding smad7 were validated with DSS-induced colitis and colitic cancer model. TRIM21 was significantly decreased in DSS-induced ulcerative colitis, whereas ameliorated colitis showed significant restoration of TRIM21

Conclusion

Leading to conclusion that loss of TRIM21 led to significant bout of IBD.
P030 Dynamics of intestinal inflammation and microbial dysbiosis in pregnant women with IBD and their infants
Authors: Tarassishin L , Kim E , Eisele C , Barre A , Thjømøe A , Mørk E , Dubinsky M , Stone J , Nair N , Debebe A , Hawkins K , Rendon A , Sabino J , Torres J , Colombel J F , Peter I , Hu J
Published: January 2020

Background

IBD often affects women during their reproductive years; however, the effect of pregnancy on disease course remains poorly understood. We aimed to assess intestinal inflammation in IBD patients compared with controls as measured by faecal calprotectin (FC). We also investigated whether maternal IBD diagnosis was associated with altered FC in the offspring and if there were particular bacterial taxa that correlated with FC levels.

Methods

Pregnant women with or without IBD and their infants were prospectively enrolled in the MECONIUM study during 2015–2018 years. FC levels at each trimester of pregnancy and in babies throughout the first 3 years of life were measured using a quantitative enzyme immunoassay (CALPRO AS, Norway). Multivariate regression analysis was applied to investigate FC levels. Stool microbiota composition in the maternal and baby stool was assessed using 16s rRNA sequencing.

Results

617 faecal samples from 342 mothers (91 IBD, 251 control) and 1005 faecal samples from 288 infants (born to 76 mothers with and 212 without IBD) were analysed. FC levels in pregnant women with IBD were significantly higher than in control pregnant women regardless of IBD type (Crohn’s disease vs. ulcerative colitis). In IBD mothers FC levels showed a decline during pregnancy (µg/g, median (interquartile range); first trimester 130.8 (102.3–147.3) vs. third trimester 85.9 (44.5–125.4), p = 0.02) (Figure 1). Patients with flare at stool collection had the highest, while those treated with anti-TNF plus thiopurine showed the lowest FC at each trimester. Babies born to mothers with IBD presented higher FC levels than those born to control mothers at multiple time points between 2 and 36 months of age (Figure 2A); the median FC levels were the highest after 1 year up to 3 years in those babies whose mothers presented active disease during pregnancy (Figure 2B). Microbial α-diversity showed an inverse relationship with FC in both mothers (r = −0.25, p = 0.032) and babies (r = −0.31, p = 0.0035). Blautia and Streptococcus abundance were positively correlated with FC in babies.

Conclusion

FC levels in IBD patients decreased throughout pregnancy. Maternal IBD, lower microbiome diversity and the abundance of certain microbial genera were associated with higher FC in the offspring up to 3 years of life. These findings suggest a potential favourable impact of pregnancy on IBD activity and highlight a possible effect of IBD during pregnancy on the intestinal inflammation in offspring, which could be mediated through altered microbiome.
P039 Regional distribution of TCR Vδ1 and TCR Vδ2 cells in healthy and inflamed mucosa of inflammatory bowel disease
Authors: Tristan E , Carrasco A , Martín-Cardona A , Zabana Y , Aceituno M , Horta D , Andújar X , Salvador I , Loras C , Fernández-Bañares F , Esteve M
Published: January 2020

Background

Vδ1+ and Vδ2+ are TCRγδ lymphocytes of the gut intraepithelial compartment that recognise proteins without help of antigen-presenting cells. Its relevance in inflammatory bowel disease (IBD) is unknown.

Aim

To measure Vδ1+ and Vδ2+ in T-cell subtypes [CD4+, CD8+, double positive (DP,CD4+CD8+), double negative (DN,CD4−CD8−) and CD103+] of healthy and IBD inflammed gut mucosa.

Methods

Twenty-six active IBD patients without immunosuppressants (n = 18 Crohn’s disease (CD), eight ulcerative colitis (UC)) and 10 healthy controls (paired biopsies of ileum, right and left colon) were included. Lymphocytes were analysed with LSRFortessa cytometer. Results expressed as % median (25–75%IQI).

Results

Healthy mucosa: reduction in ileum of total CD3+Vδ1+ due to CD3+CD8+Vδ1+ and CD3+DN_Vδ1+

Conclusion

Reduction of Vδ1+ and Vδ2+, mainly of CD103+, may play a role in IBD pathophysiology by perpetuating inflammation. Increase of CD3+Vδ1+CD4+ in both Ileal CD and UC may compensate this decrease with a selective increase in ‘helper’ function.
P042 Patient perspective on the impact of complex perianal fistulas in Crohn’s disease on quality of life: introducing the concept of the patient survey conducted in Europe
Authors: Spinelli A , Lönnfors S , Avedano L , Bojic D
Published: January 2020

Background

Perianal involvement in Crohn’s disease (CD), including fistulas, ulcers, abscesses, strictures and cancer, can lead to a significant, occasionally debilitating impairment in the patients‘ short- and long-term quality of life (QoL). Perianal fistulae can cause symptoms such as leakage, rectal pain, swelling, pain with bowel movements, and fever, which may significantly affect a patient’s social performance, sexual function and normal life activities. Fistulae are a relatively common CD complication: around 35% of CD patients have at least one fistula, while the majority of CD fistulas are complex. Still, few studies have assessed the impact of complex perianal fistulae (CPF) on the QoL of CD patients from the patient perspective.

Methods

A questionnaire assessing the effect of CPF on the QoL of CD patients was developed by patient representatives and medical experts. It consisted of 43 questions about history and activity of CD and CPF, treatment experiences, self-reported level of being informed about the disease, past and current symptoms, self-reported impact on QoL, emotions related to the disease, impact and restrictions on close relationships and social life, sexual activity, professional life and work absence. The survey was launched online on the European Federation of Crohn’s and ulcerative colitis Associations (EFCCA) website on July 15, 2019 (remains open until December 2019) and shared to national patient associations through the EFCCA network. Both CD patients with and without CPF were invited to participate in order to have a control group of patients for data analysis. The self-selective, anonymous survey was offered in English, French, German, Greek, Hebrew, Italian, Polish, Portuguese, Romanian, Spanish and Slovenian.

Results

On November 20, 2019, there were 788 responses from CD patients with and without CPF. There may have been selection bias as the survey is self-selective, only available online and in a limited amount of languages. Considering the retrospective aspect of some questions, recall bias may also have affected some of the responses. Regardless, the data collected will allow a comparison between CD patients with and without CPF, and between patients in different countries, in important, patient-relevant QoL aspects.

Conclusion

The collected data will be analysed in early 2020. The results will provide valuable insights into the often unspoken burden in the life of CD patients with CPF and potentially help in making treatment decisions for CD patients suffering from CPF.
P062 Dose-dependent differential effects of vedolizumab therapy on adhesion of regulatory and effector T cells
Authors: Becker E , Wiendl M , Schulz-Kuhnt A , Atreya I , Atreya R , Neurath M , Zundler S
Published: January 2020

Background

Vedolizumab has emerged as an important pillar of treatment in inflammatory bowel disease (IBD). However, for unknown reasons, not all patients respond to therapy. Earlier clinical studies suggested decreased response rates in the highest compared with medium dosage groups. Interestingly, vedolizumab has been shown to inhibit the homing of both regulatory (Treg) and effector T (Teff) cells and previous data from our group suggested different effect sizes in both populations. Thus, we hypothesised that the non-linear exposure–efficacy correlation might be explained by dose-dependent differential effects of vedolizumab on Treg and Teff homing. Therefore, we studied functional effects of different vedolizumab exposure levels on Treg and Teff cell trafficking.

Methods

The α4β7 expression on different human T-cell subsets as well as the binding characteristics of vedolizumab to these cells at different exposure levels was analysed via flow cytometry. Functional effects of different vedolizumab concentrations on the adhesion of Tregs and Teffs to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) were analysed using dynamic in vitro adhesion assays, transmigration assays and in vivo homing assays in a humanised mouse model. The in vivo binding of vedolizumab to Tregs and Teffs in patients receiving therapy was quantified and correlated with the corresponding serum levels.

Results

We found a preferential binding of vedolizumab to Tregs at an exposure with 0.4 µg/ml vedolizumab that shifted to a preferential binding to Teffs at an exposure with 10 µg/ml. Further increase of vedolizumab to 50 µg/ml led to equal binding to Tregs and Teffs (Figure 1). Consistently, at 10 µg/ml, dynamic adhesion of Tregs to MAdCAM-1 was increased compared with Teffs, but no difference was noted at 50 µg/ml. Additionally, a higher number of Treg compared with Teff cells were able to transmigrate in a MAdCAM-1-dependent manner at a concentration of 10 µg/ml vedolizumab. Preliminary data from homing experiments in a humanised mouse model and from IBD patients treated with vedolizumab support the notion that differential binding preferences depending on the exposure level can also be observed in vivo.

Conclusion

Our findings support a dose-dependent differential binding of vedolizumab to different T-cell subpopulations and suggest that an optimal ‘window’ of exposure exists, in which effects on Teffs predominate over Tregs. While offering a potential explanation for earlier findings in dose-ranging studies, our data might lay the basis for the establishment of individualised dose optimisation in IBD patients.
P071 Studying the effects of the probiotic bifidobacteria on gut health and intestinal epithelial cells function
Authors: Poletti M , Hautefort I , Treveil A , Demeter A , Rodriguez E , Brion A , Goldson A , Divekar D , Schofield Z , O’Neill I , Lister A , Lipscombe J , Watson A , Macaulay I , Hall L , Korcsmaros T
Published: January 2020

Background

Intestinal homeostasis depends on complex interactions between the microbiota, the intestinal epithelium and the host immune system. Paneth cells play an important role thanks to their release of antimicrobial peptides and their dysfunction has been shown to contribute to Crohn’s disease. The human gut commensal Bifidobacteria has been highlighted as a protective agent in IBD. Whilst the specific modulating factors are largely unidentified, evidence suggests that Bifidobacteria can interact with host intestinal cells. Such interactions can alter host molecular pathways, resulting in a modified intestinal cell function. Understanding these effects is crucial to unravel the potential benefits of bifidobacteria for IBD. In this study, we developed a workflow to investigate the beneficial effects of the probiotic bifidobacteria in modulating gene regulation and function of different intestinal epithelial cells in the gut.

Methods

We gavaged either conventionally raised (SPF) or germ-free mice (n = 5) with Bifidobacterium breve UCC2003 (1–5 × 108 CFU), and sorted intestinal epithelial cell types (Lgr5+ stem cells, Paneth cells and transit-amplifying cells) from the mouse intestinal tissue by flow cytometry using a panel of known antibodies. Each isolated cell population (n = 200) was subsequently profiled by low input RNA sequencing and DNA methylation.

Results

Intracellular lysozyme staining for Paneth cells and initial gene expression data exploration suggests successful separation of the different populations from the mouse intestinal tissue using flow cytometry. Gene expression data were further analysed using network biology approaches to generate cell-type-specific molecular regulatory networks indicating genes regulation by bifidobacteria in each cell types. Overlaying the methylation data will elucidate the role of epigenetics played during this regulation. Together, these networks will improve our understanding of how Bifidobacteria affects these different cells types through communication with precursor stem cells and/or with direct effects on the fully differentiated cells, ultimately impacting their function.

Conclusion

In the future, we aim to use a similar workflow using two-dimensional human-derived organoids monolayers co-cultured with bifidobacteria to investigate the effects on host intestinal epithelial cell function in a high-throughput and patient-specific manner. This will increase our understanding of the complex role played by host genetics and microbial factors in the pathogenesis of gastrointestinal diseases such as IBD, and hopefully pave the way for translational developments in the prevention these pathologies.
P072 Evaluation of dosing strategies of anti-TNFα monoclonal antibodies using pharmacokinetic modelling and simulation
Authors: Démaris A , Grišić A M , Huisinga W , Walter R , Kloft C
Published: January 2020

Background

Anti-TNFα monoclonal antibodies (mAbs) are a well-established treatment for patients with Crohn’s disease (CD). However, subtherapeutic concentrations of mAbs have been related to a loss of response during the first year of therapy1. Therefore, an appropriate dosing strategy is crucial to prevent the underexposure of mAbs for those patients. The aim of our study was to assess the impact of different dosing strategies (fixed dose or body size descriptor adapted) on drug exposure and the target concentration attainment for two different anti-TNFα mAbs: infliximab (IFX, body weight (BW)-based dosing) and certolizumab pegol (CZP, fixed dosing). For this purpose, a comprehensive pharmacokinetic (PK) simulation study was performed.

Methods

A virtual population of 1000 clinically representative CD patients was generated based on the distribution of CD patient characteristics from an in-house clinical database (n = 116). Seven dosing regimens were investigated: fixed dose and per BW, lean BW (LBW), body surface area, height, body mass index and fat-free mass. The individual body size-adjusted doses were calculated from patient generated body size descriptor values. Then, using published PK models for IFX and CZP in CD patients2,3, for each patient, 1000 concentration–time profiles were simulated to consider the typical profile of a specific patient as well as the range of possible individual profiles due to unexplained PK variability across patients. For each dosing strategy, the variability in maximum and minimum mAb concentrations (Cmax and Cmin, respectively), area under the concentration-time curve (AUC) and the per cent of patients reaching target concentration were assessed during maintenance therapy.

Results

For IFX and CZP, Cmin showed the highest variability between patients (CV ≈110% and CV ≈80%, respectively) with a similar extent across all dosing strategies. For IFX, the per cent of patients reaching the target (Cmin = 5 µg/ml) was similar across all dosing strategies (~15%). For CZP, the per cent of patients reaching the target average concentration of 17 µg/ml ranged substantially (52–71%), being the highest for LBW-adjusted dosing.

Conclusion

By using a PK simulation approach, different dosing regimen of IFX and CZP revealed the highest variability for Cmin, the most commonly used PK parameter guiding treatment decisions, independent upon dosing regimen. Our results demonstrate similar target attainment with fixed dosing of IFX compared with currently recommended BW-based dosing. For CZP, the current fixed dosing strategy leads to comparable percentage of patients reaching target as the best performing body size-adjusted dosing (66% vs. 71%, respectively).

References
P076 Role of gut microbiota in mediating the effects of thiopurines
Authors: Franzin M , Lucafò M , Lagatolla C , Stocco G , Decorti G
Published: January 2020

Background

A general consensus exists that patients with inflammatory bowel disease (IBD) present compositional changes in the gut microbiota (dysbiosis), including an increase in the abundance of Enterobacteriaceae. Thiopurine drugs are commonly used in the maintenance of remission in IBD. In this context, the purpose of the project is to explore the role of candidate bacterial strains in mediating the effects of thiopurines in vitro.

Methods

Azathioprine (AZA), mercaptopurine (MP) and thioguanine (TG) (400 µM) were incubated in minimal salts medium (M9) in presence or not of E. coli, S. enterica and K. pneumoniae and of their growth phase broths (GPB) for 4 h at 37°C. The viability of NALM6 (B cells) and JURKAT (T cells) exposed to serial dilution of drugs (ranging from 0.2 to 15 μM of AZA, from 0.3 to 20 μM of MP, from 0.08 to 5 μM of TG) previously incubated or not with bacteria and with their GPB was determined by the MTT assay. Absorbance peaks of thiopurines were analysed by UV spectrophotometry. Statistical significance was assessed by two-way ANOVA and Bonferroni’s post-test for MTT tests and by one-way ANOVA for UV spectra.

Results

In NALM6 cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 45 ± 2.9%; 34 ± 2.5%% and 21 ± 0.6%) and its GPB (respectively 41 ± 7.7%; 41 ± 5.1% and 27 ± 3.5%) compared with the drugs not previously exposed (respectively 76 ± 2.3%; 69 ± 1.7% and 43 ± 3.8%). In JURKAT cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 46 ± 2.8%; 38 ± 1.29% and 19 ± 3.3%) and its GPB (respectively 49 ± 9.4%; 38 ± 1.5% and 26 ± 1.5%) in comparison with the drugs not exposed (respectively 75 ± 4.0%; 50 ± 3.5% and 54 ± 4.0%). E. coli and S. enterica did not affect the cytotoxicity of the thiopurines. UV analysis evidenced a reduction of absorbance peaks of AZA (21 ± 0.05%), MP (32 ± 0.015%) and TG (30 ± 0.03%) after incubation with K. pneumoniae but not with its growth phase broth (GPB).

Conclusion

The activity of thiopurines decreased after incubation with both K. pneumoniae and its GPB. UV analysis suggested that the lower cytotoxicity of thiopurines exposed to the bacterial strain is due to the reduction of the concentration of the drugs exposed to K. pneumoniae. Moreover, the reduction of drug availability after the exposure to GPB could be explained with a possible interaction between thiopurines and capsular polysaccharides released by the bacteria.
P077 Metabonomic profiling distinguishes Crohn’s perianal fistulas and idiopathic idiopathic (cryptoglandular) perianal fistulas: possible clues to underlying pathogenesis?
Authors: Adegbola S , Sarafian M , Sahnan K , Tozer P , Ding N , Faiz O , Warusavitarne J , Phillips R , Holmes E , Hart A
Published: January 2020

Background

The reasons why fistulas originate have been explained in idiopathic cases, with the cryptoglandular theory being widely accepted; however, in Crohn ́s disease, it is thought to involve interplay between microbiological, immunological and genetic factors. It remains unclear why fistulas persist. Novel investigative tools in systems biology are improving our understanding of pathogenesis, and one such tool is metabonomic profiling, whereby spectrometry is used to assess metabolic responses of complex systems in health and disease. These changes are mapped using analytical and statistical techniques. To the best our knowledge, these methodologies have not been applied to aid understanding of the pathogenesis of Crohn’s perianal fistula persistence.

Methods

Fistula tissue biopsy was obtained from the fistula tract of 30 patients with idiopathic perianal fistula and 20 patients with Crohn ́s anal fistula. Two analytical platforms were attempted to achieve broad metabolome coverage. Univariate (student t-test) and multivariate statistical data analyses were performed. From the latter principle component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), models were built to find metabolites that could predict tissue samples from patients with either Crohn’s or idiopathic anal fistula. Metabolite putative identification was conducted by matching accurate mass:charge ratio (m/z) measurements of detected chromatographic features to theoretical value from in-house databases and on-line databases and previous publications.

Results

Significant OPLS-DA predictive models (validated with CV-ANOVA p-value <0.05) differentiated metabolites from tissue samples from Crohn’s vs. idiopathic anal fistula patients using both metabonomic profiling platforms (HILIC/lipid profiling). A total of 22 differentiating metabolites were identified from the HILIC profiling and 19 from the lipid profiling analysis. Mapped pathway analysis was performed for metabolites identified using online database searches.

Conclusion

Forty-one differentiating metabolites were discovered belonging to various classes of lipids, amino acids and nucleotides. Pathways involved included amino acid metabolism, phospholipid metabolism and glycerolipid metabolism amongst others. Further work in larger numbers is required to validate these findings as well as cross correlation with microbiome work to understand the impact of host gut interactions in the pathophysiology of Crohn’s and idiopathic perianal fistulas.