Introduction

Insulin-like growth factor binding protein 3 (IGFBP3) signals through the death receptor TMEM219 to modulate survival of target cells; inhibition of this signaling has been associated with a rescue of intestinal stem cell death.

Aims & Methods

Here we report the screening, generation, and characterization of fully human IgG monoclonal antibodies (mAbs) through phage display or by hybridoma technology, that block IGFBP3 or TMEM219. We used in vitro large crypts organoids (mini-guts) generated by crypts obtained from healthy subjects and from patients with inflammatory bowel disease to test the effect of the mAbs on the regenerative potentials. Expression of intestinal stem cell markers and apoptotic markers by RT-PCR was also analyzed.

Results

Both anti-IGFBP3 and anti-TMEM219 mAbs showed high affinity binding with the target antigens and potent effects in protecting self-renewal ability of intestinal stem cells in in vitro relevant assays. Among all the mAbs tested, anti-TMEM219 mAbs generated by phage display, particularly Ent001, showed the highest score in displacing the IGFBP3/TMEM219 binding and in rescuing ISC expression and function in IGFBP3-cultured human mini-guts obtained from healthy donors. In human in vitro proof-of-concept studies, in which we generated mini-guts from patients with immune-mediated intestinal disease such as Crohn’s disease, Ent001 successfully restored mini-guts growth and ISC markers’ expression, while expression of the proapoptotic IGFBP3-related factor Caspase 8 was downregulated.

Conclusion

Ent001 may represent a novel IGFBP3/TMEM219 inhibitor to be further tested and developed in in vivo models and in clinical studies as a novel therapeutic in human diseases.

Disclosure

P.F. and F.D. hold a patent on IGFBP3/TMEM219 axis. V.M., M.N., F.C. are employees of Enthera S.r.l.

Introduction

As the therapeutic armamentarium for inflammatory bowel diseases (IBD) is rapidly expanding, it is of increasing importance to optimize treatment sequencing. However, head-to-head trials comparing efficacy of vedolizumab (VDZ) in bio-naïve versus bio-experienced patients are scarce.

Aims & Methods

Aim: To compare the effectiveness of VDZ in bio-naïve versus bio-experienced patients with IBD in a real world setting.
Methods: A retrospective monocentric study was performed in IBD patients treated with VDZ at a Belgian University Hospital, from January 2018 to June 2021 with follow-up until December 2021. Primary endpoints considering efficacy after induction and after 1 year of therapy were: clinical response (CRp) and remission (CRm), biochemical response (BRp) and remission (BRm) and endoscopic response (ERp) and remission (ERm). Secondary endpoint was treatment survival during follow-up. For assessment of risk factors influencing efficacy and treatment survival, a multivariate logistic regression and Cox regression model (CRM) were applied.

Results

A total of 84 patients were treated with VDZ, 42 bio-naïve and 42 bio-experienced. At baseline, significantly more bio-experienced patients had prior intestinal resection (IR) (40.5% (17/42) vs. 16.7% (7/42), P=0.016), used an immunomodulator (IMM) (35.7% (15/42) vs. 16.7% (7/42), P=0.047) and had a longer disease duration (12.5 vs. 3.5 years, P<0.001). Bio-naïve patients had more moderate to severe comorbidities (21.4% (9/42) vs. 2.4% (1/42), P=0.007). After VDZ induction, CRp rate was higher in bio-naïve patients (60.9% (39/42) vs 39.1% (25/42), P<0.001). Multiple logistic regression analyses revealed that bio-naïve patients had a higher chance reaching CRp (aOR 14.69, 95%CI [3.12-69.14], P<0.001)(table). CRm was reached in 35.7% bio-naïve and 19% bio-experienced patients (P=0.087). No significant difference in BRp (P=0.932) or BRm (P=0.730) rate was found . A significantly higher ERp rate was seen in bio-naïve patients (90.3% (28/31) vs. 66.7% (18/27), P=0.027). Multiple logistic regression analyses showed a trend towards higher ERp rate in bio-naïve patients (aOR 4.65, [0.99-21.84], P= 0.051)(table). No significant difference was seen for ERm (32.3 % (10/31) vs. 11.1% (3/27), P=0.054). After 1 year of VDZ, CRp was significantly higher in bio-naïve patients (82.9% (34/41) vs. 62.5% (25/40), P=0.039). Multiple logistic regression analyses showed no significant difference between both groups. Univariate analyses showed significantly higher CRm rate in bio-naïve patients (70.7% (29/41) vs. 37.5% (15/40), P=0.003). After adjustment for age, disease duration, IMM use and prior IR, this difference was no longer significant. Patients without prior IR had significantly higher odds of reaching CRm (aOR 4.33,[1.13-16.59], P=0.032). No significant difference was seen for BRp (P=0.443) and BRm (P=0.313), nor for ERp (P=0.184) or ERm (P=0.061) in univariate analyses. Cox regression analysis showed significantly longer treatment survival in bio-naïve patients (aHR 2.32, [1.03-5.23], P=0.043).

Conclusion

In this tertiary real-world cohort of patients treated with VDZ, overall, a better efficacy was seen in bio-naïve compared to bio-experienced patients.

Disclosure

No conflict of interest

Introduction

Interleukin 33 (IL-33) is a pro-inflammatory cytokine that has been implicated in the pathophysiology of inflammatory bowel disease (IBD), ulcerative colitis (UC) in particular. IL-33 as a cytokine and an alarmin might evoke systemic inflammation, which could have ramifications for the functioning of the central nervous system, contributing to mood and sleep disorders. Disruptions in IL-33 concentration are well known to occur in the course of numerous psychiatric disorders as well as obstructive sleep apnea, a condition characterized by chronic low-grade inflammation and sleep fragmentation. To date, however, this protein has not been studied in relation to insomnia or poor sleep quality, frequently comorbid to chronic inflammatory and psychiatric diseases.

Aims & Methods

The study aimed to compare serum IL-33 concentrations of IBD patients and healthy controls as well as evaluate the association between the expression level of this interleukin, depression, sleep quality, and insomnia. IBD patients (n=81) and healthy controls (HC=44) were recruited. Harvey-Bradshaw index or partial Mayo were applied in the assessment of clinical disease activity for Crohn’s disease (CD) or UC respectively. Venous blood samples as well as questionnaires: Beck Depression Inventory (BDI), Epworth sleepiness scale (ESS), Athens Insomnia Scale (AIS), and Pittsburgh Sleep Quality Index (PSQI) were collected. Human IL-33 High Sensitivity Magnetic Luminex Performance Assay was used for the analysis of serum IL-33 protein concentration according to the manufacturer’s protocol. Fluorescence measurements were taken with MAGPIX, Luminex. The study has been funded by National Science Centre, Poland (2018/31/N/NZ5/03715).

Results

No differences were noted regarding IL-33 concentrations, sleep, or psychological variables between IBD patients and HC, except for sleep efficiency, which was higher in HC (p=0.004). Only in the IBD group, a positive correlation between IL-33 concentration and BDI, PSQI, and AIS (R=0.521, p=0.003; R=0.458, p=0.01; R=0.40, p=0.03), as well as negative one with sleep efficiency (R=-0.475, p=0.008) was noted. IBD individuals with high scores on ESS or PSQI (ESS>10, PSQI>5) had elevated IL-33 serum level compared to IBD patients without sleep problems (IL-33 concentration [pg/ml]: 53.385 (30.070-313.020) vs 18.795 (11.790-26.175), P=0.01 for PSQI; 270.900 (156.225-438.680) vs 21.840 (11.870-47.720), P=0.006 for ESS). IL-33 concentrations and questionnaire scores were comparable in patients with different disease activity. As for IBD types, in the CD, but not the UC group, IL-33 was positively correlated with the severity of depression symptoms and poor sleep quality (R=0.581, p=0.02; R=0.531, p=0.03) and negatively with sleep efficiency (R=-0.667, p=0.005).

Conclusion

IL-33 might be associated with mood and sleep disorders in IBD. It could exert its effect by promoting systemic and neuroinflammation, thus negatively affecting the well-being of IBD patients. Further research on the subject of changes in mental health of IBD patients in the context of anti-inflammatory or immunosuppressive treatment would be valuable.

Introduction

IL-33/ST2 axis and gut microbiota are important factors in the pathogenesis of IBD with a potential reciprocal influence. IL-33/ST2 axis controls the mucosal healing process during intestinal inflammation. Restorative proctocolectomy with ileal pouch-anal anastomosis is a surgical procedure in patients with ulcerative colitis (UC) refractory to medical therapies. Pouchitis, the most common complication, is the inflammation of the pouch with a controversial etiology.

Aims & Methods

We aimed to explore the potential role of IL-33/ST2 and gut microbiota axis in the inflammatory process of pouchitis.
30 UC patients with ileal pouch-anal anastomosis were enrolled. After clinical, endoscopic and histological assessment 18 showed pouchitis and 12 a normal pouch. Mucosal samples were collected from the afferent ileal loop and from the pouch and processed for cytokines assessment, histological evaluation and gut microbiota analyses. Multiplex analysis for inflammatory and regulatory cytokines was performed. ELISA and western blot were run to assess IL-33/ST2 protein levels and to evaluate protein isoforms, respectively. IHC was done to evaluate mucosal IL-33/ST2 expression and localization. Microbiota was analyzed by 16S ribosomal RNA gene amplicon pyrosequencing. Pouchitis Disease Activity Index and Pouchitis Histological Score were calculated, and CRP and fecal calprotectin were obtained for each patient. Multivariate analyses were run to generate transcriptional interaction networks and identify biomarkers for patients with inflamed pouches.

Results

IL-1Ra, IL-6, IL-8, IL-17, IP-10, MCP-1, MIP-1a, MIP-1b resulted increased in inflamed pouch vs. normal pouch. No differences were registered between samples obtained from the afferent ileal loop. In pouchitis, IL-7 resulted reduced in the inflamed pouch vs. the afferent ileal loop. In pouchitis, IL-33 and ST2 protein levels were reduced in the pouch vs. the afferent ileal loop and resulted overall decreased vs. patients with normal pouch indicating a potential IL-33/ST2-mediated defective healing process. Full-length, bioactive IL33, ST2L and sST2 were expressed in all experimental groups; the cleaved, less active form of IL33 was increased in only patients with pouchitis. IHC confirmed these observations. IL-33 and ST2 staining was less intense within the inflamed and ulcerated mucosa of pouchitis patients compared to normal pouch, and in close proximity to areas of re-epithelialization. An overall decreased biodiversity in patients with pouchitis vs. patients with normal pouch (both at pouch and afferent ileal loop level) was shown. Lower levels of Verrucomicrobia and increased abundance of Actinobacteria were recorded in inflamed pouch vs normal pouch. In pouchitis, the afferent ileal loop and the pouch showed significantly decreased levels of Akkermansia muciniphila and augmented abundance of Collinsella vs. samples obtained from patients with normal pouch. In pouchitis, multivariate analyses evidenced a significant inverse correlation between Collinsella e Collinsella aereofaciens abundance and ST2 levels.

Conclusion

In patients with pouchitis vs patients with normal pouch divergent inflammatory molecular pathways and microbial populations distinctly characterize the pouch and the afferent ileal loop. Overall, our results suggest a potential role for IL-33/ST2 and gut microbiota axis in driving the gut mucosal wound healing in patients with ileal pouch-anal anastomosis. Further studies are underway to determine mechanisms of action that support these findings.

Introduction

Inflammatory bowel diseases (IBD) are a distinct class of gastrointestinal disorders mainly represented by Crohn’s Disease (CD) and Ulcerative Colitis (UC). The dysregulation of microRNAs (miRNAs), a class of conserved endogenous, small non-coding RNA molecules, both tissue-derived and circulating, have shown to be promising biomarkers in IBD. These biomarkers can be useful for differential diagnosis, drug response prediction and can have prognostic value for the evolution of the disease. Moreover, miRNAs are interesting tools for potential future therapeutic approaches by miRNA antagonists or mimics [1].

Aims & Methods

In this study we aimed to perform a preliminary screening of a large panel of 179 miRNAs in the plasma of 6 patients with IBD and 12 non-IBD patients (CTRL) in order to identify a set of highly dysregulated miRNAs to be investigated in a larger cohort. Isolation of miRNAs has been conducted using miRNeasy Serum/Plasma Kit and the miRNAs expression has been evaluated using the Human serum/plasma focus, MIRCURY LNA miRNA Focus PCR panel (Qiagen) by qRT-PCR. After excluding 29 miRNAs that were not expressed in the investigated samples, we identified miR-19a-3p and miR-17-5p as the most stable miRNAs by the RefFinder analysis. Thus, the Ct values of the other 148 miRNAs have been normalized against the geometric mean of these two miRNAs as endogenous controls using the 2^-DCt method. All the clinical, sociodemographic and miRNAs expression data have been analyzed using SPSS. Categorical variables were tested by means of the chi-square test, and continuous variables with t-test or the non-parametric Man Whitney test, according to the normality of the data distribution.

Results

The two groups were age matched (mean age IBD= 45.83±21.27, mean age CTRL = 50.08±8.16, p=0.543) and did not differ for sex distribution (IBD= 33.3% female, CTRL=58.3% female, χ2= 1.00 and p=0.317). The statistical analysis revealed 45 miRNAs differentially expressed (p<0.05). In particular, we identified 29 overexpressed miRNAs (miR-223-3p, miR-143-3p, let-7f-5p, miR-30b-5p, miR-26a-5p, let-7a-5p, miR-142-3p, miR-374a-5p, miR-339-5p, miR-150-5p, miR-454-3p, let-7d-5p, miR-29a-3p, miR-146a-5p, miR-199a-3p, miR-221-3p, miR-142-5p, miR-191-5p, miR-338-3p, let-7g-5p, miR-103a-3p, miR-23a-3p, miR-145-5p, miR-374b-5p, miR-24-3p, miR-107, miR-27b-3p, let-7c-5p, miR-26b-5p) in the IBD group compared with the CTRL showing a fold regulation (FR)>1.5 and 7 miRNAs underexpressed reporting a FR<-1.5 (miR-421, miR-320d, miR-106b-3p, miR-320c, miR-20b-5p, miR-320b, miR-486-5p).
Notably, the expression of let-7a-5p that was upregulated in the IBD group (FR=2.45; p=0.001) positively correlated with the years of disease duration (p=0.01, Pearson r=0.919). The same positive correlation was observed for miR-30b-5p (p=0.049, Pearson r=0.812).

Conclusion

The reported data, showing 45 miRNAs differentially expressed in the plasma of IBD patients compared to controls, represent preliminary results that must be validated. The most statistically significant dysregulated miRNAs, as well as those that correlated with the years of disease duration and other clinical parameters will be investigated in a large IBD cohort in order to examine the potential value of these miRNAs as diagnostic and prognostic biomarkers.
This investigation received financial support from ICGEB (Contract No. CRP/22/019, ICGEB Ref. No. CRP/ROU21-01)

References

1. James JP, Riis LB, Malham M, Høgdall E, Langholz E, Nielsen BS. MicroRNA Biomarkers in IBD-Differential Diagnosis and Prediction of Colitis-Associated Cancer. Int J Mol Sci. 2020 Oct 24;21(21):7893. doi: 10.3390/ijms21217893. PMID: 33114313; PMCID: PMC7660644.

Introduction

Nowadays, Non-Alcoholic Fatty Liver Disease (NAFLD) is a leading cause of liver disease and liver transplantation. Although obesity is a well-recognized risk factor for NAFLD, there are data to support that not all patients with NAFLD are necessarily obese. However, the evidence on the prevalence and risk factors for NAFLD in lean individuals with Inflammatory Bowel Disease (IBD) is scarce.

Aims & Methods

The aim of the study was to assess the prevalence and risk factors for NAFLD in lean population with IBD. This is a cross-sectional, case control study including lean individuals with IBD (cases) and lean individuals without IBD (controls), matched for age and sex. Patients with risky alcohol consumption, previous diagnosis of chronic liver disease (immune, viral, genetic) and secondary causes of NAFLD were excluded. All included patients were performed a liver ultrasound, transient elastography and laboratory tests. Lean individuals were defined as Body Mass Index < 25 kg/m², and NAFLD as ultrasound steatosis and/or Controlled Attenuation Paremeter >248 dB/m. A descriptive analysis was carried out, and the relationship between variables was assessed using Chi square test/Fisher´s test (categorical variables) or T-student/Mann-Whitney U test (quantitative variables), as appropiate. In addition, a multivariate analysis was performed, presented as adjusted Odds Ratio (OR) and 95% Confidence Interval (95%CI).

Results

A total of 443 participants, 363 lean cases and 80 lean controls, were included in the study. No differences were observed in terms of age, sex and metabolic risk factors between both groups (Table 1). The prevalence of NAFLD in lean individuals with IBD was significantly higher compared to lean individuals without NAFLD (22% vs. 10%, p=0.015). Multivariate analysis confirmed that IBD was an independent risk factor for NAFLD in lean population (OR 2.75 95%CI (1.18-6.41)). In addition, male sex (OR 1.78 95%CI (1.04-3.05)) and older age (OR 1.03 95%CI (1.01-1.05)) were identified as risk factors for NAFLD in lean individuals.

Table 1. Baseline characteristics of cases and controls.

Conclusion

The prevalence of NAFLD is significantly higher in lean population with IBD, suggesting that IBD could play a role in the development of NAFLD in lean individuals.

Disclosure

This project has received funding from “Diputación General de Aragón” (code: DGACOVID-02).

Introduction

Being two immune-mediated diseases (IMIDs), the association between multiple sclerosis (MS) and inflammatory bowel disease (IBD) is plausible, but data in the literature are conflicting.

Aims & Methods

The aim of our study was to evaluate the possible association between IBD and MS in a cohort of patients with IBD.

Results

In a retrospective study, we examined the medical records of 5,739 patients with a confirmed diagnosis of IBD followed in our clinic between 1978 and 2022.
Among these patients, we identified 14 with MS, with a prevalence of 0.24%. The reported prevalence of MS in general population in northern Italy in 2021 was 0.18% (P = 0.24). For each of the patients with MS identified, more than ten patients without MS were analyzed. The 14 MS cases were then compared with 342 controls. From the 14 MS patients, 12 (85.7%) were female and 2 (14.3%) were male, while in the control group, 158 (46.2%) were female and 184 (53.8%) were male (P = 0.004). As for therapy, significant differences were found on mesalazine (5 (41.7%) cases vs. 317 (92.7%) controls, P < 0.0001) and anti-TNF treatment (0% cases vs 26.6% controls, P = 0.03, respectively) at the time of MS diagnosis. Moreover, Kaplan-Meier curve analysis showed that the 20-year survival probability was 98.4% for patients with IBD, while for patients diagnosed with MS and IBD it was 82.1% (P = 0.02).

Conclusion

In conclusion, patients with IBD have a similar risk of developing MS compared to the general population, but female sex appears to increase the risk. Indeed, life expectancy at 20 years for patients with IBD and MS is lower than for patients with IBD alone.