Introduction

Inflammatory Bowel Disease (IBD) is characterized by epithelial barrier injury of the gastrointestinal (GI) tract. The disease course encompasses abnormal immune responses and excessive secretion of proteases from immune cells. Neutrophils are the first to migrate into the inflamed interstitial matrix, where type III collagen is significantly deposited. Detection of mucosal inflammation in early stages is crucial to prevent cumulative clinical damage, as a delayed diagnosis can hinder effective treatment. Thus, we aimed to develop a biomarker that reflects early intestinal inflammation prior to it becoming medically evident; allowing us to distinguish patients that would respond to an anti-inflammatory treatment.

Aims & Methods

A competitive enzyme-linked immunosorbent assay targeting a human neutrophil elastase derived fragment of type III collagen was developed (C3-HNE). The validation was conducted by assessing its dilution recovery, matrix spiking, interference, and inter- and intra-assay variation. The biological relevance was evaluated in commercial samples, one preclinical, and two clinical studies. In the preclinical study, C3-HNE was measured in serum samples from a dextran sodium sulfate (DSS)-induced colitis rat model. In the commercial samples and clinical cohorts, C3-HNE was measured in serum from patients with IBD and healthy donors (HD). To compare the biomarker levels between groups, two-way ANOVA with an uncorrected Fisher's LSD, and Mann-Whitney U-tests or Kruskal-Wallis (Dunn’s corrected) were conducted.

Results

The novel biomarker C3-HNE was successfully validated, resulting in a technically robust assay with acceptable technical parameters (Table 1). In both clinical cohorts and commercial samples, C3-HNE levels were significantly higher in patients with Crohn's disease (CD), ulcerative colitis (UC) and IBD than in HD (p<0.01, p<0.05 and p<0.05) (Table 1). In the preclinical study, the analysis revealed significant difference in the C3-HNE levels between the control and DSS groups on day 4 (p=0.02). Additionally, C3-HNE was released earlier (day 1) in the timeline than the elevation of the current disease activity (DAI) score (day 7) (Table 1).


Table 1. Technical performance characteristics of the assay C3-HNE and C3-HNE levels in a DSS-induced colitis model, commercial samples and two cohorts. For the validation the acceptance criteria were 100 ± 20 % and <15% for inter-intra variation. For the biological relevance that was assessed in healthy and diseased (CD, UC and IBD) donors of commercial samples and cohorts I and II, data is presented as medians with IQR. For the DSS-induced colitis model, two-way anova test and uncorrected Fisher's LSD results are presented. The bold values are meant to highlight the significant difference.

Conclusion

C3-HNE is elevated in patients with CD, UC and IBD compared with HD. Furthermore, C3-HNE reflects early stages of clinically apparent mucosal damage during experimental colitis. This biomarker holds the potential to identify early mucosal damage or acute inflammation in the GI tract; nevertheless, additional studies are needed to evaluate its clinical validity.

Disclosure

Morten Asser Karsdal, Anne-Christine Bay-Jensen, Joachim H. Mortensen are full-time employed and stock owners at Nordic Bioscience.
Marta S. Alexdottir and Martin Pehrsson are full-time employed at Nordic Bioscience.

Introduction

Irritable bowel syndrome (IBS) is defined as a disorder of gut-brain interaction (DGBI) and is characterised by intestinal immune activation, increased intestinal permeability, and structural and functional brain alterations. DGBI patients have a higher prevalence of psychological comorbidities. Nevertheless, the exact pathophysiological link between these factors and the sequence of events is unknown. Gut alterations have been suggested to induce brain neuroinflammation in DGBI, but scientific evidence is lacking.

Aims & Methods

The present study aims to assess whether GI alteration induces brain neuroinflammation, alters brain function, and, consequently, induces psychological comorbidities. A unique longitudinal study was conducted from 30 to 220 days of age to establish the chronological sequence of intestinal and neurological changes. For this, we utilised the BioBreeding diabetes-prone (BBDP) rat model, which spontaneously develops intestinal inflammation, increased permeability, and colonic hypersensitivity^{1, 2}and has been employed as a model of DGBI. The BioBreeding diabetes-resistant (BBDR) was used as control. Anxiety-like behaviour was assessed using the elevated plus maze. Intestinal permeability was assessed in vivo by measuring the passage of 4 kDa and 70 kDa dextrans into the blood, thereby evaluating paracellular and transcellular permeability, respectively. The appearance of red colour, given by the mixture of the dextrans in the stools, was used to assess overall intestinal motility. The blood-brain barrier (BBB) integrity and brain metabolic profile were assessed using contrast-enhanced magnetic resonance imaging (0.25 mL/100g weight of 0.05 mM, gadolinium) and proton magnetic resonance spectroscopy, respectively.

Results

BBDP rats exhibit an increased colonic permeability starting from 90 days, and reduced GI motility at 90 and 160 days. Interestingly, this increased permeability is accompanied by an increased BBB permeability at 220 days only in the anterior cingulate cortex (ACC) but not in the hippocampus, insula, amygdala, or thalamus. BBDP rats showed a slight increase in total choline and myoinositol concentrations in the ACC and the insula at late time points, which may indicate increased neuroinflammation. BBDP rats also have higher concentrations of glutamine-glutamate in the insula and N-acetyl aspartate in the ACC. Moreover, BBDP rats develop anxiety-like behaviour at 160 and 220 days.

Conclusion

The BBDP rat model supports the current hypothesis that increased gut permeability can lead to brain neuroinflammation, altered function, and, consequently, to psychological comorbidities.
EU-funded DISCOvERIE project (No. 848228).

References

1. Meleine M, Accarie A, Wauters L, et al. Colonic hypersensitivity and low-grade inflammation in a spontaneous animal model for functional gastrointestinal disorders. Neurogastroenterol Motil 2019;31:e13614.
2. Vanuytsel T, Vanormelingen C, Vanheel H, et al. From intestinal permeability to dysmotility: the biobreeding rat as a model for functional gastrointestinal disorders. PLoS One 2014;9:e111132.

Introduction

Pneumococcal vaccination is essential in IBD patients. However, its efficacy in patients on immunosuppressive or anti-TNF therapy is much lower than in the normal population (45% vs 80%). Vaccine regimen recommendations vary according to the degree of immunodepression. The aim of our study was to identify an optimal vaccination strategy for immunocompromised IBD patients treated with anti-TNF, vedolizumab and/or IS.

Aims & Methods

This is a prospective, multicenter, randomized study comparing two vaccine regimens: intensified vaccine strategy (Prevenar13®/Prevenar13®/PSV-23® (or Pneumovax as a replacement for PSV-23) vs. normal strategy (Prevenar13®/PSV-23® (or Pneumovax as a replacement for PSV-23), (randomization 1: 1) on vaccine immunogenicity measured by antibody titer against at least 9 of the 13 pneumococcal serotypes contained in Prevenar13®. Any patient with confirmed IBD under biotherapy (anti TNF with or without immunosuppressive drugs or Vedolizumab) and in clinical remission (HBI <4 for CD and partial Mayo Score <3 for UC) was included. Vaccination and vaccine monitoring were carried out by the vaccination center of each center, with adverse events reported at each visit (8 for 36 months of follow-up), and ELISA and OPA serotype assays systematically collected and clinical activity was reported by the gastroenterologist at the same time. Correlate of protection in adults using serology testing was classically defined as at least 9 out of 13. Serotypes > 1µg/mL. For functional testing of antibodies using the Opsonophagocytic assays, we apply the well-defined 4 times increase of titer between pre and post-vaccination against at least 70% of serotypes. ClinicalTrials.gov Identifier: NCT02255227

Results

51 patients in the normal vaccination arm and 53 in the intensified vaccination arm were included (mean age: 42.9 years, male: 63%, CD: 68%, active smoking: 21%, duration of IBD: 10.2 years, 98% IBD inactive). Around 88% of patients were under anti-TNF monotherapy (28% on combotherapy) and 12% under vedolizumab. Serology criteria for good responders at M5 were 57.5% for the intensified regimen vs 25.6% for the standard regimen (RR: 2.25 (1.26;4.00), p = 0.003). For OPA correlate of protection, vaccine response was significantly greater in the intensified arm at all-time points: M5 (96% vs. 89%), M12 (76 vs. 58%) and M36 (68% vs. 36.8%). In multivariate analysis, two factors reduced vaccine response: anti-TNF use (RR: 6.84; CI95: 1.01-46.1; p=0.048) and CD type (RR: 9.48; CI95: 2.74-32.8; p:0.0004). The tolerability of the vaccine regimens was comparable throughout follow-up. In terms of IBD activity, percentage of clinical relapse was similar during follow-up (16.8% under the intensified regimen versus 25% under the normal regimen (p:NS).

Conclusion

In IBD on biotherapy and/or IS, the intensified pneumococcal vaccine regimen (2 injections of Prevenar followed by one of Pneumo 23) is significantly more immunogenic than a standard regimen, especially when using functional correlate of protection. The duration of the response is also greater in this intensified arm.