bluuebunny/crossref_metadata_2025_split huggingface dataset:
P027 Anorectal Motility Disorders in Inflammatory Bowel Disease patients
Authors: Costache R , Dimitriu A , Les A , Gheorghe C 
Published: January 2023

Abstract

Background
In spite of prolonged disease remission of IBD patients, induced by the new biological molecules, a significant number of them suffer from persistent debilitating symptoms with major impact on the quality of life. Frequently, these symptoms are due to post-inflammatory motility changes and misinterpreted as functional disorders. Our aim is to identify and characterize the anorectal motility dysfunction in IBD patients.

Methods
We are conducting an ongoing prospective study started in August 2019, which includes the IBD patients admitted in a Tertiary Gastroenterology Centre in Bucharest, with specific symptoms (anorectal pain, incontinence, difficult defecation). We perform high resolution anorectal manometry using Sandhill Scientific systems, the parameters being analysed using InSIGHT software. The manometric testing comprise measurements of anorectal pressure at rest, during squeeze, simulated evacuation, rectoanal inhibitory reflex (RAIR) and rectal sensory testing, in compliance with International Anorectal Physiology Working Group protocol.

Results
We studied 21 patients (12 patients with Ulcerative Colitis and 9 patients with Crohn’s Disease, 15 females and 6 males, mean age 40 (±11.43) years. Only 23.1% (5 patients) had rectal active involvement. Symptoms were reported by 81.0% (17) patients: pain (57.1%), anal incontinence (94.1%), difficult evacuation (29.4%), urgency (64.7%) and intolerance of rectal therapies (35.3%); rectal inflammation was not correlated with the presence of symptoms in our study group (p= 0.53). Modified manometric parameters were found in 81.0% patients and were associated with previous pelvic surgical interventions (p&lt;0.05); although, the latter does not seem to increase the risk of incontinence (p=0.33). In 61.9% cases the manometric measurements correlated with the symptoms. 85% of the patients with passive incontinence presented lower resting pressure and 57.1% of those with active incontinence were found with lower squeeze pressures. Changes compatible with dyssynergia were detected in 61.9% of the cases. Sensory testing revealed alterations in 46.2% of patients, and RAIR was negative in 33.3% of the cases.

Conclusion
There is a considerable number of patients with anorectal motility changes. Therefore, pelvic floor investigation is an essential tool in the management of IBD patients with anorectal symptoms.
P033 The assessment of serum cytokines at baseline could predict mucosal healing in patients with Crohn’s disease treated with ustekinumab. A prospective study.
Authors: Bertani L , Antonioli L , Fornili M , D’Antongiovanni V , Coppini F , Ceccarelli L , Carmisciano L , Benvenuti L , Mumolo M G , Bottari A , Baglietto L , Bellini M , Fornai M , De Bortoli N , Costa F 
Published: January 2023

Abstract

Background
Ustekinumab (UST) is a monoclonal IgG1 antibody binding the p40 subunit of IL-12 and IL-23. The blockage of the activation of the heterodimer IL-12/IL-23 inhibits the differentiation of Th1 and Th17 cells, reducing the inflammatory cascade typical of Crohn’s disease (CD). Some patients with CD achieve mucosal healing after one year of UST therapy, whereas others experience primary non-response or loss of response. No biomarkers are currently available to predict therapeutic response to UST. The aim of this prospective study was to identify one or more biomarkers able to predict mucosal healing in patients with CD treated with UST.

Methods
We prospectively enrolled all consecutive CD patients treated with UST, following the current guidelines. A blood sample was drawn before UST infusion at week 0 (baseline), 24 and 48, and serum cytokines IL1α, IL-1β, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-22, IL-23 and IL-33 were evaluated by a fluorescence assay. At the same timepoints, Fecal Calprotectin (FC) was assessed. A colonoscopy was performed before starting the treatment and at week 48, where therapeutic outcome was evaluated in terms of mucosal healing, defined as a Simple Endoscopic Score for CD &lt;2.

Results
Out of 44 patients enrolled, 22 (50%) achieve mucosal healing at the end of follow up. Baseline IL-23 levels were higher in responders than in non-responders (Mann-Whitney test, p-value&lt;0.001), whereas higher baseline levels of IL-1α are associated with non-response (p=0.03). We also observed a higher probability of mucosal healing in those with lower levels of IL-6 at baseline (p=0.05). A borderline statistically significant association was observed for baseline FC, which values were lower in responders than in non-responders (Mann-Whitney test, p=0.098). No significant differences were observed with regard to the baseline levels of the other cytokines. FC levels decreased over time in responders, whereas its values did not change over time in non-responders (test for the interaction between time and outcome, p&lt;0.001). IL-13 presented a homogenously decreasing time trend regardless the outcomes (test for the association with time and outcome, p=0.019). No significant time trend was observed with regard to the other cytokines.

Conclusion
Baseline serum levels of IL-23, IL-1α and IL-6, as well as the decrease of FC over time, showed a good reliability in predicting therapeutic outcome to UST in CD in terms of mucosal healing. These biomarkers could help clinicians in the perspective of a better management of UST therapy, identifying early the patients with higher probability to respond to the treatment.
P040 Caspase-8 blockade attenuates NSAID-induced colitis and cell death via NLRP3 inflammasome and NF-κB activation
Authors: Singh R , Rossini V , Stockdale S , Draper L , Hill C , Nally K , Shanahan F , Andersson-Engels S , Melgar PhD S 
Published: January 2023

Abstract

Background
Non-steroidal anti-inflammatory drugs (NSAIDs) are believed to exacerbate symptoms and inflammation in patients with inflammatory bowel disease (IBD). Recent studies indicate the gut microbiota may play a role in NSAID-promoted inflammation. However, mechanism(s) underpinning NSAID-induced inflammation are not fully understood. In this study, using the piroxicam-accelerated model of colitis (PAC) in IL-10-/- mice, we examined the mechanism(s) regulating NSAID-induced colitis.

Methods
To establish whether microbiota plays a role in PAC, IL-10-/- mice were treated with streptomycin for 24hrs followed by feeding with 100 ppm piroxicam in their food for 9 days. In the inhibitor study, mice were injected intra-peritoneally with the caspase-8 inhibitor (Z-IETD-FMK, 10mg/kg) every other day starting before piroxicam-feeding. Colon tissue was collected on day 14 for analysis on inflammation and cell death markers by qRT-PCR, MesoScaleDiscovery (MSD)-assay, western blot, and histology. Faecal samples were collected at different time-points for 16S rRNA gene sequencing.

Results
A significantly higher expression of colonic inflammatory markers and a reduced expression of epithelial barrier proteins and elongated crypts were found in PAC-mice, which was independent of microbiota. Piroxicam feeding did not cause major changes in fecal microbiota diversity or composition on day 14. An activation of members of the NLRP3 inflammasome family including caspase-1 and IL-1b, Caspase-8, and the pro-apoptotic markers -cleaved caspase 3 and PARP- were associated with piroxicam-induced colitis. Inhibition of caspase-8 ameliorated NSAID-provoked colitis and apoptosis by preventing mitochondrial damage, and downregulating NF-κB and NLRP3 activation as well as reducing cleaved caspase-3 and PARP levels. Histology analysis revealed an improvement in colonic epithelial morphology associated with shorter crypts and reduced immune cell infiltration to the lamina propria.

Conclusion
Our data provides evidence on how NSAIDs can promote intestinal inflammation by activating caspase-8, NLRP3 inflammasome and NF-κB, resulting in induction of inflammation and apoptosis. Targeting Caspase-8 could be a potential treatment strategy for patients with NSAID-worsened symptoms and inflammation
P047 Osteopontin in colitis-associated carcinoma (CAC)
Authors: Manna S , Sehn M , Cardoso da Silva D , Heldt C , Horstkorte L , Elezkurtaj S , Siegmund B , Weixler B , Hummel M , Gröne J , Schumann M 
Published: January 2023

Abstract

Background
Inflammatory bowel disease (IBD) patients pose a lifelong risk of developing colitis-associated carcinoma (CAC). Current understanding and mechanisms of CAC development stem from murine CAC models while studies on human CAC carcinogenesis are scarce. Thus, we aimed to understand CAC carcinogenesis, using human samples from patients suffering from Crohn′s disease (CD), CD-associated CAC (CD-CAC), ulcerative colitis (UC), and UC-associated CAC (UC-CAC) and further validated the findings in vitro.

Methods
We obtained FFPE specimens from 40 patients that suffered from either one of the IBDs or CACs and furthermore inflammation-free healthy controls (10 patients per group). These samples were micro-dissected, followed by RNA isolation and Nanostring gene expression analysis. Our data revealed a strong upregulation of SPP1 (Secreted Phosphoprotein-1) gene encoding for osteopontin (OPN) upon comparing heathy controls to IBD and to CAC. We then used in vitro-models to elucidate the mechanism of OPN-induced carcinogenesis. We stimulated intestinal epithelial cell lines HT-29/B6 and T84 cells with rhOPN (recombinant human OPN), following bulk RNAseq. We validated the findings using Seahorse assay for analysis of mitochondrial respiration (determining oxygen consumption rate, OCR). Western phospho-blots were done to unravel signal transduction events.

Results
Nanostring gene expression data revealed the following: (i) SPP1 gene encoding for OPN and epithelial-to-mesenchymal (EMT) transcription factors fibronectin-1 (FN1) and ZEB1 were strongly upregulated in UC-CAC and CD-CAC patients when compared to UC and CD. (ii) IL17-pathway genes were significantly downregulated in UC-CAC patients. (iii) Tight junction and polarity genes were altered in both UC-CAC and CD-CAC patients. rhOPN stimulation in vitro to HT29/B6 and T84 cells excluded OPN as the trigger of EMT but uncovered upregulated mitochondrial respiratory chain genes as a potential trigger for tumour cell proliferation. Validation of RNAseq findings by Seahorse assay revealed that after sequential injections of oligomycin, FCCP, rotenone and antimycin A both, HT-29/B6 and T84 cells, had higher basal and maximal OCRs in response to rhOPN (Fig. 1). T84, but not HT-29/B6 cells also showed an upregulated ATP production in response to rhOPN.

Conclusion
SPP1 expression correlates with CAC carcinogenesis. Putative underlying pathways rather include modulation of mitochondrial respiratory chain activity by differential gene expression than induction of EMT.
P053 Ustekinumab decreases circulating Th1, Th17, and MAIT cells in Crohn’s disease
Authors: Lord J , Konecny A , Shows D 
Published: January 2023

Abstract

Background
Ustekinumab is a humanized antibody to a p40 subunit shared by the cytokines IL-12 and IL-23, which has been approved for the treatment of Crohn’s disease. While it is presumed to derive clinical efficacy by blocking the ability of these cytokines to stimulate specific T cell subsets, its actual effect upon the human immune system has not been well-described.

Methods
Peripheral blood mononuclear cells (PBMC) were cryopreserved from 20 Crohn’s patients before and during ustekinumab therapy. Thawed PBMC were then analyzed by flow cytometry for cell surface markers and, after overnight stimulation, intracellular cytokines. Success (n=9) or failure (n=11) of ustekinumab therapy was at the discretion of the treating physician, blinded to flow cytometry data.

Results
A significant decrease in IL-17A-producing Th17 (p=0.001) and CD161+/TCRVα7.2+ MAIT (p=0.005) cells was seen overall in response to ustekinumab. While no significant effect was seen overall in IFN-γ -producing CD4 (Th1) and CD8 T cells, there was a dichotomy between treatment responders and non-responders, with Th1 cells decreasing in responders (p=0.004) and increasing in non-responders (p=0.03) after ustekinumab initiation. IFN-γ+ CD8 T cells likewise increased in non-responders (p=0.01), although their decrease in responders was less significant (p=0.12). While there was no difference in the pre-treatment frequency of Th17’s or IFN-γ+ T cells between responders and non-responders, MAIT cells were significantly more common in the blood of responders (p=0.03).

Conclusion
While the IL-23-blocking effect of ustekinumab on Th17 and MAIT cells was seen across all recipients, its IL-12-blocking effect on IFN-γ-producing T cells was restricted to treatment-responsive patients, suggesting that its mechanism of action may differ from newer, IL-23-specific agents. Whether the frequency of MAIT cells before or a rise in IFN-γ-producing T cells during therapy can predict treatment failure warrants additional study.
P061 Response to anti-α4β7 therapy in Ulcerative colitis is associated with lymphoid aggregate attrition through impaired recruitment of naïve lymphocytes
Authors: Canales-Herrerias P , Uzzan M , Seki A , Czepielewski R S , Verstockt B , Dunn A , Dai D , Ko H M , Furtado G C , Tokuyama M , Tankelevich M , Meringer H , Cossarini F , Jha D , Paulsen J D , Nakadar M Z , Wong J , Sharma K , Rosenstein A , Dawson T , Singh H , Lim J , Lira S A , Yajnik V , Reboldi A , Petralia F , Vermeire S , Polydorides A D , Randolph G J , Colombel J F , Mehandru S 
Published: January 2023

Abstract

Background
Vedolizumab (VDZ) is a frontline drug for Ulcerative colitis (UC) and Crohn’s disease (CD) that targets integrin α4β7, a gut-homing receptor. Despite significant use, the mechanism(s) of action (MOA) of VDZ remain unclear.

Methods
Peripheral blood mononuclear cells (PBMC) from UC patients (n=43) and paired intestinal biopsies in a subset (n=12) were examined at week 0 (pre-infusion) and 14, by multiparameter flow cytometry (FC). TNF inhibitor (TNFi)-treated UC patients served as controls. Drug MOA was further examined in detail in murine models using single-cell RNA-seq, FC, conventional and immunofluorescent microscopy (IF). Photoconvertible (Kikumi) mice were used to study cellular trafficking after anti-α4β7 (DATK32) antibody (mAb) administration. Two distinct UC cohorts (n=42 and n=21 respectively) were used to correlate GI immune alterations following VDZ with mucosal healing.

Results
A significant decrease of colonic and ileal naïve B and T cells was noted in VDZ- but not in TNFi-treated patients (Fig 1A), while total T cell-frequencies were unchanged. Circulating gut-homing plasmablasts (β7+) were significantly decreased post-VDZ.
Peyer’s patches (PP) in anti-α4β7-treated mice showed a rapid loss of cellularity associated with decreased follicular naïve B cells (Fig 1B). Single-cell RNA-seq also demonstrated a significant loss of follicular B cells, including a unique population of epithelium-associated B cells following anti-α4β7 mAb. In Kikumi mice, anti-α4β7 mAb impaired non-photoconverted follicular B cells and T cells into photoconverted PPs (Fig 1C) demonstrating that loss of PP cellularity was due to impaired cellular ingress.
In VDZ-treated (but not TNFi-treated) UC patients, lymphoid aggregate (LA) size was significantly reduced in treatment responders compared to non-responders (defined by absence of histological inflammation). A distinct validation cohort further confirmed that reduced lymphoid aggregate size was associated with response to VDZ-therapy (Fig 1D).

Conclusion
VDZ is associated with impaired ingress of naïve B and T cells, resulting in attrition of intestinal LA. Reduced LA size is associated with VDZ response. Immune inductive site targeting represents a novel MOA of VDZ in patients with UC.
P063 Host-mycobiome interactions during the resolution of inflammation in experimental colitis
Authors: De Salvo C , Wargo H , Vidmar K , Di Martino L , Mahabaleshwar G , Cominelli F , Ghannoum M , Pizarro T 
Published: January 2023

Abstract

Background
Recent studies have documented the complexity of the intestinal fungal community (‘mycobiome’) in mice, and clinical and experimental observations have shown that the mycobiome influences both gut health and disease, e.g., IBD. In fact, prior studies have shown that CD patients, compared to healthy controls, harbor higher levels of intestinal Candida tropicalis (Ct), which is the major fungal species detected in the colons of colitic mice after dextran sodium sulfate (DSS) challenge. Moreover, proteins encoded by genes within IBD susceptibility loci, such as the pattern recognition receptor (PRR), NOD2, are known not only to recognize bacterial components, but also a fungal cell wall element, chitin. In fact, the role of PRRs in regulating immunity against intestinal fungi, and how fungi influence IBD remains poorly defined.

Methods
We challenged DSS colitic wildtype (WT) and Nod2-/- mice with Ct 2 days before DSS administration and subsequently on day 0, 3 and 6. Bone marrow-derived macrophages (BMDMs) from untreated mice were administered chitin in vitro.

Results
Our data confirms previous studies that Ct challenge does not exacerbate colitis in DSS-treated C57BL/6 WT mice, however, Ct-infected Nod2-/- mice possess a higher fungal burden and exhibit worse colitis symptoms, such as weight loss, decreased stool consistency, and presence of blood in stools, vs. Ct-infected WT mice, indicating an essential and protective role for NOD2 during colitis recovery after Ct challenge. Our results also show that Ct-infected Nod2-/- mice display a marked reduction in colonic Il22 and Il17, which are cytokines previously reported to be important in maintaining epithelial barrier integrity during DSS colitis. These data were confirmed in colons of Ct infected DSS challenged, ileitis prone SAMP1/YitFc (SAMP) mice NOD2 deficient. Moreover, our in vitro data show, a decrease in Il1ß and Il23 in BMDM from SAMP Nod2-/- mice compared to WT after 2h of exposure to chitin. IL-17+ innate lymphoid cells (ILCs) are also known to control fungal burden during opportunistic fungal infections, and interestingly, our findings indicate that Ct infection of Nod2-/- vs. WT mice results in a decreased frequency of mesenteric lymph node–derived type 3 ILCs (ILC3s), suggesting that delay in fungal clearance and recovery in Nod2-/-mice may be due to the inability to mount protective type 3 immune responses.

Conclusion
Taken together, the data collected suggests that NOD2 is essential to maintain gut mycobiome homeostasis and drives protective innate immune responses, via a macrophage-mediated ILC3 recruitment and IL-17 mechanism, by preventing the overgrowth of opportunistic fungi that may contribute to chronic intestinal inflammation, such as that observed in CD.
P072 Circadian misalignment by simulated night shifts decreases colonoid barrier integrity in Ulcerative Colitis
Authors: Swanson G , Shaikh M , Keshavarzian A , Scheer F 
Published: January 2023

Abstract

Background
Circadian rhythms orchestrate multiple biological processes with 24-hour oscillations that regulate diverse bodily functions. The central clock is in the suprachiasmatic nucleus and orchestrates peripheral circadian clocks in every organ system including the gastrointestinal tract (GIT). Previous work has shown that circadian misalignment in Ulcerative Colitis (UC) is associated with increased colonic permeability, and colonic organoids recapitulate the host circadian phenotype. The goals of this study were to examine whether circadian misalignment would disrupt the colonic barrier integrity in UC.

Methods
5 subjects, 2 UC and 3 Healthy Controls (HC), were recruited into the study. All UC subjects were inactive (partial Mayo Score ≤ 1) and stable medications with no flares for the last 3 months. For two weeks prior to entry into the circadian lab all subjects were on a prescribed regular sleep schedule. Subjects were then kept in the circadian lab for 6 days with strict control of their light/dark cycle (Figure 1). All subjects then underwent two unprepped flexible sigmoidoscopies at: (1) baseline with circadian alignment and (2) after three days of simulated night shifts with circadian misalignment. Colonic organoids generated created from the colon tissue biopsies grown in Matrigel and barrier integrity was assessed in apical-out colonoids by FITC-Dextran (4 kDa) Net Fluorescence was estimated by ImageJ.

Results
Colonic organoids at baseline had significantly decreased ZO-1 in UC versus HC, Figure 2. At baseline there was an increase in UC colonic organoid barrier integrity as measured by net fluorescence (density/area). In UC vs HC barrier integrity was 855 ± 203.8 vs 333.1 ± 52.9, p&lt;0.05. After three days of simulated night shifts, barrier integrity significantly increased in both groups at was 1100 ± 342 vs 656 ± 197.9,p &lt;0.5 (Figure 3). After 24 hours of treatment with cytokines, barrier integrity in HC, UC, misaligned HC, and misaligned UC was 2227 ± 376, 2030 ± 264, 1787 ± 402, and 1506 ± 332, (Figure 4).

Conclusion
Circadian misalignment by alternation of light:dark cycles (simulated night shift) causes misalignment of host central clock and impacts circadian regulated biological process like tight junction proteins which decreases colonic barrier integrity. This study highlights the importance of studying environmental factors that impact circadian timing in UC including night shift work which may be a significant risk factor for a disease flare. Further in vivo and in vitro studies are ongoing to determine the mechanisms through which circadian misalignment impacts colonic barrier integrity in UC (NCT05180279).