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Organoids are self-renewing, 3D structures, consisting of different cell types, with histology and physiology features very close to the physiology of the studied organ. Specifically, human Intestinal Organoids (HIOs) develop epithelial crypts consisting of all subtypes of intestinal epithelial cells which are surrounded by mesenchymal cells. Our aim was to develop 3D HIOs from human embryonic stem cells (hESCs) and examine the expression of fibrotic and mesenchymal factors during their maturation process. Additionally, we investigated the effect of the pro-inflammatory cytokines, IL-1α and TNF-α on the expression of fibrotic and inflammatory mediators in HIOs.

The human ESC line (H1) was cultured and then differentiated towards HIOs using commercially available kit. HIOs were characterized by immunofluorescence in all differentiation stages. In order to examine their maturation process, we compared the mRNA expression of fibrotic and mesenchymal markers from passages 1-10. In order to examine their functionality, HIOs from different passages were stimulated with 5ng/ml IL-1α and 50ng/ml TNF-α for 12 hours, total RNA was collected and the fibrotic and inflammatory mRNA expression was examined. The mRNA transcripts of CD90, collagen type I, III, fibronectin, CXCL8, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR.

HIOs were successfully developed as they were stained positive for all tested markers throughout their developmental process. Regarding their maturation process, we observed high expression of CD90, collagen type I, type III and fibronectin that was gradually decreased during passages. As for the fibrotic and inflammatory responses from HIOs, we found that the IL-1α and TNF-α stimulation resulted in statistically significant upregulation of the fibrotic factors, fibronectin, collagen type I and type III in culture passages 2 and 4, but had no effect in culture passages 8 and 10. Similarly, IL-1α and TNF-α stimulation led to the statistically significant induction of the inflammatory chemokines CXCL8, CXCL10 and CXCL11 in culture passages 2 and 4, while no effect was observed in culture passages 8 and 10.

Our findings indicate that HIOs contain a functional mesenchymal component that is gradually diminished during passages. Inflammatory and fibrotic responses of HIOs seem to depend on the fitness of their mesenchyme. IBD studies using HIOs as in vitro models should be performed on early passages, when HIO’s mesenchymal component is still functional.

Accurately predicting disease course at diagnosis is critical to facilitate personalized therapy in inflammatory bowel disease (IBD). PredictSURE IBD^TM is a whole blood qPCR assay that was developed to predict prognosis in newly diagnosed, treatment-naïve IBD patients – classifying them into IBDhi (high-risk) or IBDlo (low-risk). The current recommendation is that PredictSURE IBD^TM should not be used in those who have commenced steroids. In this study, we aimed to determine the impact of steroid therapy on the performance of PredictSURE IBD^TM .

Whole blood was serially taken from patients admitted with severe IBD requiring intravenous (IV) steroids (pre-steroid, day 3, day 5; n=10, cohort 1) and from patients receiving oral steroids as outpatients (pre-steroid, week 1, week 6; n=10, cohort 2). An independent cohort of 43 IBD patients, all within 3 months of diagnosis and on corticosteroid treatment (41 systemic and 2 topical, cohort 3) was recruited. RNA was extracted and analyzed with PredictSURE IBD^TM (PredictImmune, UK). Patients were prospectively followed and treated according to routine clinical management by physicians blinded to the test results, and clinically stratified according to one of the original definitions used to construct and validate the test (need for step up to immunosuppressive or biological therapy or surgery).

In cohorts 1 and 2, both oral and intravenous steroids affected the PredictSURE IBD^TM result: misclassification as IBDlo occurred in 5/8 IBDhi patients receiving oral, and 5/7 IBDhi patients receiving IV, steroids. In 60% this change was detectable early (within 1 week of oral steroids and 3 days of IV steroids). Steroids did not affect the classification of IBDlo patients. Consistently, the prognostic accuracy was limited in patients already receiving steroids (cohort 3). After a median follow-up of 31.8 [IQR 18.7 - 42.1] months, 35 (81%) patients required step-up therapy. PredictSure IBD^TM correctly classified only 23 (54%) patients with accuracy of 0.53 (sensitivity: 0.51, specificity: 0.63, positive likelihood ratio: 1.38, negative likelihood ratio: 0.77). Seventeen (80%) of the misclassifications were clinically high-risk patients who were predicted as IBDlo. Time to treatment escalation was similar between patients classified as IBDhi or IBDlo after starting steroid therapy (p= 0.47) (Figure 1).

The prognostic accuracy of PredictSURE IBDTM is limited if performed after steroid therapy has begun, most likely because of the misclassification of high-risk patients as low risk. Therefore, the test should only be performed in patients with active disease who are not receiving steroid therapy, as currently recommended.

Local mesenchymal stromal cell (MSC)-therapy is approved for the treatment of Crohn’s disease-associated perianal fistulas. However, little is known about the working mechanism of local MSC-therapy. For the first time we evaluated engraftment and immunoregulatory effects of local MSC-therapy in patients with refractory proctitis. To do so, we analyzed biopsies and serum from patients with ulcerative proctitis before and after treatment with endoscopically injected MSCs in a phase IIa clinical trial (EudraCT number 2017-003524-75).

Thirteen therapy-refractory ulcerative proctitis patients were endoscopically injected bone marrow-derived allogeneic MSCs from healthy donors. Clinical efficacy was evaluated by the endoscopic and full Mayo score. Engraftment of the MSCs was investigated using fluorescence in-situ hybridization (FISH) of sex chromosomes on post-treatment biopsies. The presence of anti-HLA-antibodies against the MSC-donor was determined in the serum. Changes in immune cell subsets were evaluated using cytometry-by-time-of-flight (CyTOF) analysis.

Thirteen patients with an endoscopic Mayo score of 2 (n=3) or 3 (n=10) of the rectum were treated with local MSC-therapy. Although complete remission was not achieved, full Mayo score was improved at week 6 (median 8 [IQR 6-10]) compared to baseline (median 11 [IQR 9.5-12]) (p=0.001). Preliminary data using FISH on the Y-chromosome, indicated the presence of MSCs in the rectum biopsies of female patients treated with male donor derived-MSCs at week 6. At baseline, HLA-antibodies were present in four patients. Six weeks after local injection of the MSCs, two out of thirteen patients developed new class I and II HLA-antibodies against the MSCs. Interestingly, in two patients pre-existing HLA-antibodies showed increased/boosted levels after local MSC-therapy, while one additionally developed new HLA-antibodies. CyTOF analysis of inflamed rectal biopsies 6 weeks after MSC treatment revealed significantly increased frequencies of several myeloid subsets (i.e. CD11b+CD14+CCR7+/-CD127+CD25+HLADR+ and CD14+HLA-DR-CD123-CCR7+) and a subset of CD4+ memory T cells with a more exhausted/regulated phenotype (PD-1+TIGIT+CD69+CD38+CD69).

Local MSC-therapy in patients with refractory proctitis changed the rectal immune profile characterised by a significant increase in a subset of effector memory CD4+ cells and several myeloid subsets, which might be associated with immune modulation. These results provide the basis for future studies on the mechanism of action of MSCs on rectal mucosa. New anti-HLA class antibodies developed in 2/13 patients after local administration. Whether these latter results have consequences for MSC-donor selection deserves further study.

Measuring food-related quality of life (FRQoL) quantifies the psychosocial impact of eating and drinking.¹ The influences on FRQoL in people with inflammatory bowel disease (IBD) are not well explored, despite IBD being a chronic disease affecting the digestive tract. This study aimed to characterise and identify any patient or disease-related predictors of FRQoL in individuals with IBD.

Adults with a formal diagnosis of IBD were recruited to a prospective multi-centre cross-sectional study between April 2018 and December 2019. Participants completed questionnaires measuring FRQoL (IBD-FRQoL-29: minimum/poor 29, maximum/greatest 145), clinical disease activity (active disease: Harvey Bradshaw Index >4 active disease, Simple Clinical Colitis Activity Index >2, restrictive eating behaviour (Nine Item Avoidant/Restrictive Screen: minimum 0, maximum 45), mental health (DASS-21: minimum 0, maximum 126) and other patient and disease-related variables.

One hundred and eight participants completed the questionnaires. The majority of the cohort had UC (69/108, 64%) and there was almost equal distribution of those with quiescent (48%) and active (52%) disease The mean FRQoL of individuals with IBD was 79 (95% CI 75, 84) (see Figure 1). Poorer FRQoL was seen in those with restrictive eating behaviour associated with fear of a negative consequence from eating (p<0.0001) and reduced appetite (p<0.030). Greater FRQoL was seen in those with lower disease activity (p<0.0001) and previous IBD surgery (p=0.240). FRQoL was not influenced either way by IBD phenotype, duration, or gender. The majority of participants obtained their dietary information from the internet (60%) or gastroenterologist (46%).

FRQoL in people with IBD is poorer in those with restrictive eating behaviours and clinically active disease.  Interestingly, it was greater in those with previous IBD surgery. Further research is required to validate these associations and explore longitudinal effects of poor FRQoL on patient outcomes and potential strategies for prevention or management of impaired FRQoL in IBD.


References
¹Hughes LD, King L, Morgan M, et al. Food-related quality of life in inflammatory bowel disease: Development and validation of a questionnaire. J Crohns Colitis 2016;10:194-201.

The prevalence and risk of Eating Disorders (ED) in IBD, despite the potential overlap of these two conditions, have been rarely reported. ED diagnosis should be considered in patients with IBD and multidisciplinary approach would be recommended in these complex cases to provide an adequate therapeutic intervention. Screening tools to evaluate eating attitudes and behaviours in patients with IBD could be used in daily practice, as for example the Eating Attitude Test – 26

Children and adolescents (8-18 years) with IBD and age and gender matched healthy controls were prospectively enrolled in 5 italian pediatric IBD units between June 2019 and August 2020. Subjects with an existing diagnosis of ED were excluded. The risk of ED was assessed using a 26 points Likert scale screening tool (CH-EAT-26 and EAT-26 for children < and > 14 years respectively), with a total score of 20 or above indicating a risk for ED. Correlations between clinical and disease’s parameters and the CH-EAT-26/EAT-26 score were calculated

110 patients with IBD and 110 age and matched healthy controls were screened with the CH-EAT26/EAT-26 questionnaire. The total EAT26 scores and the prevalence of an at-risk score (score>20) did not differ in IBD subjects compared to controls. IBD patients were more frequently on an exclusion diet with lactose free-diet being the most common regimen. Furthermore, 8.1% of IBD children was on a partial enteral nutrition (PEN). In IBD subjects elevated scores on the Ch-EAT26/EAT-26 were associated with being younger (r=-0,2226, p=0.002), following an exclusion diet (r=0.25, p=0.009) and a partial enteral nutrition (PEN: r=0,2507, p=0.009). Type, duration and activity of disease, gender, weight, height and BMI z-scores were not significantly correlated to the CHEAT26/EAT-26 score. Being on a PEN  and  following an exclusion diet were the only independents factors influencing the EAT26 score at the multiple regression analysis (p= 0,004; p= 0,034; R2 = 0,25)

Our results indicate that 5.45% of IBD children have a behavior at risk for developing an ED, a percentage that is not statistically different compared to healthy controls. A particular follow-up should be reserved to patients on restricted diets and on partial enteral nutrition, that can develop maladaptive attitudes toward eating. The development of a disease specific tool or a validation of pre-existing questionnaires would help to identify a robust screening instrument and ultimately to correctly classify the risk of patients. Once the risk is correctly assessed it is mandatory to address the patient to a specific multidisciplinary follow-up.

Recent progress in deciphering the complex pathogenesis of Crohn’s disease (CD) has yielded several effective biologicals. However, ambitious therapeutic goals remain unfulfilled as almost 30% of patients are primary non-responders to a particular biological. This underscores the need for easy-to-implement biomarkers that predict (non-)remission. We aimed to identify serum protein biomarkers that predict endoscopic remission in CD patients.

Serum samples from 169 consecutive CD patients with active endoscopic disease (presence of ulcerations) before starting a biological [infliximab (IFX), adalimumab (ADA), vedolizumab (VDZ) or ustekinumab (UST)] to which they were naïve were collected. Patients were prospectively followed with endoscopic re-assessment after 6-12 months. There were 102 patients (Table 1) with endoscopic remission (SES ≤ 2 or disappearance of all ulcers), whereas 67 showed no improvement. Two independent and complementary proteomic platforms were used: 644 proteins belonging to predesigned assays were quantified using Proximity Extension Assay (PEA) technology (Olink Proteomics AB, Sweden). Second, wide protein discovery mass spectrometry (MS)-based technic (Caprion, Canada) was used and quantified another 985 proteins. A multivariate modelling framework was then applied on a randomly selected training sub-cohort (85%). Predictive performance of identified panels was assessed on the remaining test sub-cohort (15%). We sought to implement the same framework on the drug-specific subgroups; however, train/test splitting was not possible in IFX or ADA subgroups due to very few observations in the non-remission arms which diminishes the possibility for reliable predictive modelling.

Applying the modelling framework on training sets from the general cohort, VDZ subgroup and UST subgroup, proteomic panels were selected and consisted of 26, 6 and 8 proteins, respectively, and showed high performance in the test sets (Table 2).  VDZ and UST panels shared only 2 proteins each with the general panel, and had no predictive power (accuracy ≤ 0.5) when used to predict other subgroups, making them specific to their respective drugs. Selected proteins are involved among others in pro-inflammatory, extracellular matrix modelling, coagulation and cellular-vascular interaction pathways (Table 3).

Applying a multivariate machine learning algorithm on a wide pool of serum proteomics analysed through two discovery technics, we were able to identify 3 proteomic panels that can predict endoscopic (non)remission in patients with CD. Exact implication of these proteins in intestinal inflammation and a validation in an independent cohort is being further investigated.

Ulcerative Colitis (UC) associated single nucleotide polymorphisms (SNP) are mostly in non-coding regions of the genome. Because of that, it has been challenging to determine their role in the disease onset and severity. We have previously developed an integrative workflow (termed iSNP) to understand better how these SNPs are involved in the pathogenesis of UC. Here we present a recent update both in the methodology and new results, including a new player for prediction of therapeutic escalation in UC.

From immunochip data of 376 UC patients of an East-Anglian, UK cohort, the SNPs were filtered for only the UC-associated ones. Then we predicted the SNPs’ effect on regulatory interactions using two complementary transcription factor-target gene prediction methods, RSAT and FIMO. SNPs were considered if the SNP was located in the promoter region of a gene or in an enhancer region of a gene defined by the HEDD database. We considered a gene ‘SNP-affected’ if the risk allele and the non-risk allele had different transcription factor binding sites detected by any of the two methods. The proteins encoded by the SNP-affected genes were mapped to the integrated and high-confidence signaling network resource OmniPath. We also identified the direct physical interactors (first-neighbours) of these SNP affected genes/proteins. We created networks for each patient separately using their individual SNP-profiles. Finally, based on these patient-specific networks, we clustered patients in an unsupervised manner.

We found 15 UC-associated SNPs which affected transcription factor binding sites, which in turn were modulating 54 genes. From these 54 SNP affected genes, 29 coded proteins that were present in the OmniPath signaling network. The patients formed five clusters, which were significantly correlated with therapeutic escalation defined by mesalazine or other more advanced therapy (p <0.05). Patients requiring immunomodulatory treatment have a greater prevalence of  SNP RS943072 (G), corresponding to the transcriptional regulation of VEGF (vascular endothelial growth factor). VEGF is elevated in UC and stimulates angiogenesis, which is involved both in tissue regeneration and inflammation. VEGF is upregulated in the presence of this risk SNP causing increased inflammatory phenotype.

We updated the iSNP method by including enhancer regions and multiple transcription factor binding site prediction methods, and were able to predict that those UC patients who have a VEGF-affecting SNP require therapeutic upscaling.

α4β7 blockade is a well-established therapy in ulcerative colitis (UC), acting in part by preventing lymphocyte ingress into the mucosa. The β7 unit of the α4β7 heterodimer is shared by α_Εβ7, which is expressed on both tissue resident memory cells and γδ intra-epithelial lymphocytes (IEL). It was hypothesised that targeting both α4- and α_Εβ7 might be more efficacious; however mixed results from phase III studies of β7 blockade asks questions of the biological relevance of different α_Eβ7 expressing cells.

Colonic biopsies were obtained during endoscopy from >40 subjects. Lymphocytes were isolated using short term culture or digested from whole tissue. RNA sequencing was performed on α_Εβ7^pos and α_Εβ7^neg colonic γδ T cells from 4 donors and findings were validated by flow cytometry.

α_Εβ7 is widely expressed on TCRαβ CD8 cells and γδ IEL in both non-IBD controls and the uninflamed mucosa in UC, but its expression is significantly reduced on analogous subsets harvested from inflamed UC. On further study, the capacity of TCRαβ CD8 T cells to make TNFα and IFNγ on stimulation is similar between α_Εβ7^pos and α_Εβ7^neg cells, whereas in the γδ T cell compartment α_Εβ7^neg cells produce significantly more pro-inflammatory cytokine than their homeostatic α_Εβ7^pos counterparts. To examine the cells’ biology further, γδ T cells were isolated according to their α_Εβ7 status and RNAseq undertaken. This revealed a distinct signature with α_Εβ7^neg cells demonstrating an activated phenotype high in markers such as CD18, CD5 and lymphoid homing receptor CCR7 whereas α_Εβ7^pos cells demonstrate a homeostatic tissue-resident phenotype, expressing immune checkpoints TIGIT and CD101 and gut-homing marker CCR9. On culturing tissue from non-IBD controls in pro-inflammatory cytokines, IL-12 and IL-18, the γδ T cell compartment down-regulated α_Εβ7 and TIGIT and upregulated CD18, in part recapitulating a disease phenotype. On examining previously affected mucosa of patients who have achieved mucosal healing, α_Eβ7 expression of the γδ T cells returned to a profile resembling non-IBD controls whereas the expression levels in the inflamed mucosa remained predictably low.

This study demonstrates that α_Εβ7 expression is low in active UC but restored in mucosal healing. α_Εβ7^neg cells are proinflammatory with a distinct phenotype which may in part be recapitulated by inflammatory cytokines in vitro; whereas α_Εβ7^pos cells demonstrate a homeostatic phenotype, which may both reflect and maintain steady state barrier integrity. Hence in terms of γδ T cells, pharmacological β7 blockade has potential to interfere with the homeostatic roles of α_Εβ7 expressing cells while having little effect on a potentially pathogenic subset of tissue α_Εβ7^neg γδ cells.

The efficacy and safety of risankizumab (RZB) as induction and maintenance therapy forpatients with moderately to severely active Crohn’s disease (CD) has been documented. Steroid-free clinical remission is an important additional treatment goal in CD. The efficacy of RZB by baseline steroid use during induction and steroid-free outcomes during maintenance was examined.

In ADVANCE (NCT03105128) and MOTIVATE (NCT03104413), patients with moderately to severely active CD received intravenous (IV) RZB induction therapy or placebo (PBO) for 12 weeks. Patients with clinical response to RZB were re-randomised in a 52-week maintenance study (FORTIFY; NCT03105102) to subcutaneous (SC) RZB or PBO (ie, withdrawal). Patients receiving steroids (maximum prednisone or equivalent ≤ 20 mg/day; budesonide ≤ 9 mg/day) at baseline of the induction studies maintained stable doses for the 12-week study duration. A mandatory steroid taper per protocol was initiated at the beginning of maintenance for patients receiving steroids during induction. Patients losing clinical response (per investigator assessment) after initiation of taper could have their steroid dose increased up to the induction baseline dose. This analysis included patients who received RZB 600 mg IV in ADVANCE or MOTIVATE and patients who received RZB 360 mg SC in FORTIFY. Endpoints reported included clinical remission (defined by CD Activity Index [CDAI] or stool frequency/abdominal pain score [SF/APS] criteria) at week 12 of induction by baseline steroid use, steroid-free clinical remission (CDAI or SF/APS), steroid-free endoscopic response, and steroid-free endoscopic remission at week 52 of maintenance. Steroid discontinuation rates over 52 weeks of maintenance were also assessed.

Induction of clinical remission at week 12 with RZB 600 mg IV in ADVANCE or MOTIVATE was independent from baseline steroid use (Figure 1).Clinical remission rates (CDAI or SF/APS) at week 12 were similar for patients using steroids vs those who were not (33.8%–42.0% vs 34.9%–46.6%; Figure 1). Steroid use decreased over time in FORTIFY, with a greater proportion of patients receiving RZB 360 mg SC discontinuing steroids at week 52 vs withdrawal (PBO SC; Figure 2A). Rates of steroid-free clinical remission (P ≤ .012), steroid-free endoscopic response (P < .001), and steroid-free endoscopic remission (P < .001) were significantly higher with RZB 360 mg SC vs withdrawal (PBO SC) at week 52 of maintenance (Figure 2B–2C).

Efficacy of RZB induction therapy was independent of baseline steroid use. RZB maintenance promotes high rates of steroid-free clinical and endoscopic outcomes, demonstrating a benefit of RZB treatment in CD.