Introduction

Bowel preparation is recognized as the most burdensome aspect of the whole colonoscopy procedure, sometimes even worse that the examination itself, and represents a major barrier to screening colonoscopy. Mannitol could significantly improve patient acceptability as it is rapid, requires a single dose, a low volume and has a pleasant taste. The SATISFACTION study compared oral mannitol 100g/750ml with standard split-dose 2L PEG-ASC (MoviPrep®).

Aims & Methods

The SATISFACTION Phase III study was an international, multicentre, randomized (1:1), parallel-group, endoscopist-blinded non inferiority trial. Primary endpoint was the proportion of patients with adequate bowel cleansing. Secondary endpoints concerned efficacy (adenoma detection rate, caecal intubation rate, time of evacuation), safety (intestinal gases concentration, haematobiological parameters, adverse events) and patient satisfaction. The study included 703 patients (352 treated with mannitol and 351 with PEG-ASC) undergoing elective colonoscopy.

Results

Mannitol was not inferior to PEG-ASC for proportion of patients with adequate cleansing (91.1% and 95.5% respectively; p value for the non-inferiority =0.0095). There was no significant difference for secondary efficacy endpoints. The profile of acceptability was significantly better in mannitol group for easy to use, taste, willingness to use (p<0.0001 for all parameters). The concentration of intestinal gases (H₂, CH₄) was similar in the two groups and well below those potentially critical.

Conclusion

Mannitol was not inferior to PEG-ASC for proportion of patients with adequate cleansing (91.1% and 95.5% respectively; p value for the non-inferiority =0.0095). There was no significant difference for secondary efficacy endpoints. The profile of acceptability was significantly better in mannitol group for easy to use, taste, willingness to use (p<0.0001 for all parameters). The concentration of intestinal gases (H₂, CH₄) was similar in the two groups and well below those potentially critical.

Disclosure

Prof. Gian Eugenio Tontini and Prof. Maurizio Vecchi received a consultant fee from NTC Pharma. Anna Orsatti is a current NTC Pharma employee

Introduction

Motorized spiral enteroscopy (MSE) has shown to be safe and effective for deep small bowel enteroscopy in patients with and without prior abdominal surgery, including patients with Crohn’s disease.
This case series reports our first results regarding the feasibility and safety of the MSE with endoscopic balloon dilatation in patients with small bowel strictures due to Crohn’s disease.

Aims & Methods

Data were collected retrospectively on patients who underwent MSE for endoscopic balloon dilatation of small bowel strictures due to Crohn’s disease. Strictures were either postoperative (P.O.) or post-inflammatory (P.I.).

Results

A total of ten consecutive patients with Crohn’s disease underwent 13 procedures. Propofol sedation without endotracheal intubation was used in all procedures. Demographic and procedure related data are shown in the table. One patient underwent three procedures with 3–4 month intervals. During the third procedure triamcinolone acetonide was injected at four quadrants of the stricture. Fluorescence imaging was used during six procedures. The procedure was technically successful in 12/13 of procedures. In one case, dilatation was not performed because of a longer than anticipated and a sharply angulated stricture.
The number of treated strictures ranged from one to five per procedure. Dilatation of multiple strictures in two patients (4 and 5) was achieved within a relatively short procedure duration of 55 and 70 minutes. Two retained video capsules were retrieved.
Besides the anticipated mild abdominal discomfort and/or sore throat in three patients, no adverse events occurred.

Conclusion

These retrospective data show that MSE with CRE dilatation is feasible and safe in a cohort of patients with strictures due to Crohn’s disease, postoperatively as well as post-inflammatory. The fact that the distal 16 cm of the endoscope are not covered by the spiral overtube makes cautious introduction of the endoscope beyond a recently dilated stricture feasible and safe. Therefore, dilatation of multiple strictures in one session is possible, provided that they are at a short distance from each other. Fluorescence imaging guidance may be helpful in severe stenosis, multiple strictures, or sharp angulations.

Introduction

Crohn's disease is characterised by accumulation of immune cells in the intestinal mucosa. This includes effector T cells, thought to play key roles in disease through cytotoxic activity and pro-inflammatory cytokine production. The CCR9-CCL25 axis is the dominant chemokine system that mediates recruitment of mononuclear cells into small bowel mucosa. We sought to develop a novel humanized anti–CCR9 mAb with enhanced effector function (AZD7798) that potently depletes circulating and tissue resident CCR9 expressing cells.

Aims & Methods

Mice were immunized with CCR9 overexpressing cells and CCR9 hybridomas binders were selected. A lead clone was then humanised and optimised via CDR mutagenesis. Cell binding assays was used to identify specific binders to human and cynomolgus CCR9, and antibody-dependent cell-mediated cytotoxicity (ADCC) was confirmed in Molt4 as well as primary human PBMCs. CCR9 expressing cells in human peripheral blood as well a human gut tissue were characterised by flow cytometry. Finally, CCR9 cell depletion flow cytometric assays were established to investigate circulating CCR9 cell depletion in non-human primates (Macaca fascicularis) at a dose of 10 mg/kg, and ileal depletion was assessed by immunohistochemistry.

Results

8,448 hybridoma were screened and 10 unique hits were identified. The AZD7798 parent antibody was humanised and optimised leading to 40 fold potency improvement. AZD7798 demonstrated specific binding to human and cynomolgus, but not murine, CCR9. In the absence of NK cells, AZD7798 prevented binding of CCL25 to CCR9, and in the presence of NK cells induced potent ADCC in an in vitro human PBMC assay. We showed that CCR9 is highly expressed in human intestinal tissue, with highest levels in the ileum (small intestine). Memory T cells, B cells and T regs accounted for the majority of intestinal CCR9 expression, with minimal expression on other immune populations. CCR9-expressing CD4+ and CD8+ T cells showed increased pro-inflammatory cytokine production compared to CCR9-negative T cells, demonstrating their pathogenic potential. Finally, in cynomolgus monkey, AZD7798 depleted circulating and ileal CCR9 expressing T cells, achieving complete depletion in blood within 2 days.

Conclusion

AZD7798 delivers potent blood and tissue depletion of CCR9 expressing mononuclear cells in-vivo, in non-human primates. Safety of AZD7798 is currently being assessed in Ph1, clin trial registry number NCT05452304. This might provide a novel treatment approach for small bowel inflammatory diseases, including Crohn’s disease.

Disclosure

All authors are employees/shareholders of AstraZeneca

Introduction

Ulcerative colitis (UC) is a refractory inflammatory bowel disease of the colon with repeated relapses and remissions that requires lifelong treatment. Previous studies indicated that proinflammatory cytokines involved the onsets and developments of UC. New treatments for targeting these cytokines, such as TNF-α, IL-12 and IL-23 have been developed in a recent decade, underlying that immune abnormalities are the core for the etiology of UC.
Platelets play a role not only in hemostatic coagulation but in inflammatory mediator by interacting with various cells in the innate and adaptive immunity. Chronic inflammatory response confers platelets activation leading to elevated platelet count and hypercoagulability. Moreover, platelets aggregates with leukocyte in active inflammatory milieu. In fact, thrombocytosis, thromboembolism and upregulation of platelet-monocyte complex (PMC) in UC patients have been reported, implying that platelets could be novel biomarker, therapeutic target and clues for elucidating the pathology of UC. However, the clinical significance of platelet activation and PMC formation in UC is not fully elucidated and few studies were conducted to assess how platelet activation contributes to disease aggravations of UC. Therefore, we comprehensively analyzed platelet activation and platelet-monocyte complex by focusing on a correlation with clinical severity of UC.

Aims & Methods

Patients with UC (n=70) including MES0-2 group (n=34) and MES3 group (n=27), diagnosed according to a combination of symptoms, endoscopic findings, histologic findings, and the absence of alternative diagnoses, and age and sex-matched healthy controls (HCs) (n =19) were enrolled for this study. We analyzed the surface markers such as CD62P, CD40L, CD63, PAC-1 and Annexin V on CD61+ platelets by flowcytometry (FCM) in patients with UC in comparison with those in healthy adults. We also analyzed the proportion of CD61+CD14+ platelet-monocyte complexes and quantified the activation of PMCs by FCM. Moreover, we also investigated the correlation among surface markers, proportion of platelet-monocyte aggregates and Mayo endoscopic score (MES) in UC patients.

Results

The expression of CD62P but not other surface markers on platelets was significantly upregulated in UC when compared to HCs (p<0.01), and was significantly upregulated in in MES3 group when compared to the MES0-2 group (P<0.001). The proportion of PMC was also higher in MES3 groups when compared to MES0-2 group (P<0.05), and was markedly reduced by treatment (P<0.05). In addition, CD16+ monocytes, phenotypically mature and pro-inflammatory cell population, were increased in patients with UC when compared to HCs (P<0.01). The expression of CD16 on monocytes aggregated with platelets was significantly higher when compered to that without platelet (P<0.001).

Conclusion

Our study suggests that platelet activation accelerates mucosal inflammation via adhesion with monocytes in UC and contributes to the aggravation of disease.

References

Semple JW, et al. Platelets and the immune continuum.
Nat Rev Immunol. 2011 Apr;11(4):264-74.
Collins CE, et al. Platelets circulate in an activated state in inflammatory bowel disease.
Gastroenterology. 1994 Apr;106(4):840-5.

Introduction

Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are non-infectious chronic inflammatory disorders of the gastro-intestinal tract. The continuous presence of inflammatory stimuli, such as increased circulation of cytokines and danger-associated molecular patterns (DAMPs), leads to an enhanced immune response in these patients that could explain some types of disease relapse. An important receptor for DAMPS and pathogen-associated molecular patterns (PAMPs) is NLRP3, a cytosolic pattern recognition receptor. The NLRP3 inflammasome is exclusively activated by inflammatory stimuli, like DAMPs and PAMPs, and therefore not expressed without inflammation. It is thought that NLRP3 associates with ASC to recruit pro-Caspase 1 to the NLRP3 inflammasome and cleave it to its active form. In turn, Caspase 1 cleaves pro-interleukin 1 beta (IL-1β) and pro-interleukin 18 (IL-18) to their mature forms. IL-1β is a known elevated pro-inflammatory cytokine in IBD, promoting intestinal inflammation and immune cell recruitment. We hypothesized that the NLRP3 inflammasome pathway is therefore upregulated in IBD patients.

Aims & Methods

We first set out to systematically analyze gene expression of NLRP3 and IL1B in IBD patients by performing a systematic literature review and subsequent meta-analysis according to the PRISMA guidelines and Cochrane Collaboration methods. Subsequently, an observational cohort of 70 active and quiescent patients with CD or UC was sampled for whole blood, to study NLRP3 inflammasome gene regulation, protein production, assembly and secretion. We performed ex vivo LPS and ATP challenges of whole blood, with and without NLRP3 inflammasome formation inhibitor MCC950.

Results

Across thirty studies with publicly available mRNA expression data, we found NLRP3 and IL1B expression to be significantly higher in IBD patients than in non-IBD controls. In fresh IBD patient-derived whole blood, LPS and ATP stimulation activated the NLRP3-related genes, protein production and subsequent cytokine secretion. No IL-1β and IL-18 were secreted upon incubation with MCC950, suggesting a crucial role for NLRP3 inflammasome assembly in IL-1β and IL-18 production in immune cells. In patients with active IBD, the inflammasome was transcribed at a higher level than in patients in remission. However, this effect was lost on the protein level. We found a large spread in IL-1β and IL-18 production, suggesting a variation in activation of the NLRP3 inflammasome between individuals which was not related to disease activity.

Conclusion

NLRP3 and IL1B mRNA transcription is significantly increased in IBD patients compared to non-IBD controls, and significantly higher in patients with active disease than in patients in remission. While this fits the inflammatory state of active IBD, we found that the level of IL-1β and IL-18 production by the NLRP3 inflammasome is not significantly different between disease states. This suggests a variance in individual immune response to challenges, that could help identify a subset of IBD patients that would benefit from additional therapies directed at NLRP3 and IL-1β.

Introduction

Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial aetiology, which is still not completely understood. OCTN1, an organic cation transporter could have a role in modulating the inflammatory response, whose genetic polymorphisms were associated to increased risk of inflammatory bowel diseases. Up to now, limited information exists on its role in predicting/modulating response to therapies.

Aims & Methods

The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and immune composition to Infliximab therapy in murine colitis. Dextran sodium sulphate model of colitis on C57BL/6 mice and ocnt1 gene knockout mice was used to assess clinical efficacy of infliximab administered intravenously. Stool, colon and mesenteric lymph node samples were collected to assess differences in gut microbiota composition, histology for Rachmilewitz score and T cell populations respectively.

Results

Octn1^-/-influences microbiota profile and is associated with a worse dysbiosis in colitis mice. Infliximab treatment attenuates colitis-associated dysbiosis with increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1^-/- mice have a higher baseline percentage of Treg, Tmemory, Th2 and Th17.

Conclusion

Our data support murine model to study OCTN1 genetic asset in inflammatory bowel diseases. This could be the first step to consider this membrane transporter as a biomarker in inflammatory conditions and as a predictor of response to therapies.

Introduction

Inflammatory bowel disease (IBD) is characterized by chronic inflammation due to an uncontrolled immune response orchestrated through, i.e. neutrophils. The basement membrane acts as a front-line defense promoting tissue integrity, where the α-3 chain of type IV collagen is solely expressed on the outermost layer of the mucosa. Detection of subclinical ulceration is crucial since early-stage IBD presents a unique window of opportunity for intervention, as the disease might become a self-sustaining process once deep inflammation becomes established. We therefore, wanted to develop a biomarker reflecting early tissue injury, aiding in the implementation of prompt treatment.

Aims & Methods

A competitive enzyme-linked immunosorbent assay quantifying a human neutrophil elastase (HNE) degraded fragment of type IV collagen α-3 chain was developed (C4A3-HNE). Dilution- and spiking recovery, inter- and intra-variability, and interference were evaluated. Acceptance criteria for technical validation were a recovery of 100% ±25 and an inter-intra variability of <15%. Commercial serum samples were purchased from ProteoGenex. Biological relevance was validated in an observational study from Odense University Hospital (OUH) and healthy controls. Groups were compared using Kruskal Wallis (Dunn‘s corrected).

Results

The technical validation of the C4A3-HNE assay demonstrated acceptable results for recovery, variation, and interference. Biological relevance was assessed in commercial IBD samples and healthy donors. C4A3-HNE was elevated in both CD and UC compared to healthy (p=0.001 and <0.0001)(AUC [95% CI]: 0.820 [0.669-0.971]; 0.875 [0.775-1.000]). The results were validated in an independent cohort of IBD patients (both p<0.0001). For UC, analysis showed that compared to patients in endoscopic remission, C4A3-HNE is elevated in patients with mild endoscopic activity, decreasing dose-dependently for moderate and severe disease (T1). Fecal calprotectin and CRP levels were also lower in IBD patients with high levels of C4A3-HNE (T1). With a cut-off value of 54.1 ng/ml, C4A3-HNE could significantly discriminate between mild and severe endoscopic disease (T1).

Conclusion

Our study demonstrates the biological and possible clinical relevance of the novel C4A3-HNE biomarker. The biomarker is elevated in patients with IBD compared with healthy, and high levels may reflect early mucosal damage in patients with UC. Furthermore, inflammatory markers were lower in IBD patients with high levels of C4A3-HNE, pointing towards high levels being indicative of early disease. C4A3-HNE may provide early detection of mucosal damage or prediction of relapse; however, further studies are required to assess the clinical validity of this biomarker.

Disclosure

MA, MP, MK, and JM are employees of Nordic Bioscience A/S. MK owns stocks in Nordic Bioscience A/S.