Introduction

Endoscopic and histologic remission are key therapeutic targets in Ulcerative Colitis (UC). Recently, intestinal barrier healing has demonstrated superiority in predicting clinical adverse outcomes. It is promising, especially in patients with primary sclerosing cholangitis (PSC) colitis, which has more aggressive disease behaviour and a higher risk of colorectal cancer.¹ Hence, novel real-time and in-vivo tools for the intestinal barrier are pivotal.

Aims & Methods

This study aimed to assess intestinal barrier healing in UC using ultra-high magnification endocytoscope (ECS) and automated multispectral imaging. Thirty-four UC patients, 68% with PSC, in clinical remission undergoing surveillance colonoscopy, and 3 healthy controls were prospectively enrolled from two tertiary university referral centres. Ileal, ascending and descending colonic mucosa were evaluated with high-definition, virtual chromoendoscopy, and ECS, with biopsies taken from each segment. Endoscopic remission was defined as MES ≤1, UCEIS≤1, PICaSSO≤3, while histological remission as RHI≤3 without lamina propria or epithelial neutrophils, NHI ≤1 and PHRI=0. A novel ECS score was applied to assess ileal and colonic intestinal barrier (Table 1). Furthermore, the barrier was deeply characterised through automated multiplex immunofluorescence (inForm® Akoya Biosciences), quantifying tight junctions (TJs). Specifically, the cell density and mean expression of Claudin-2, Occludin and JAM-A were assessed in all intestinal segments. Patients were followed up for a median of twelve months, and adverse outcomes, including flare-ups, hospitalisation, colectomy and changes in medication, were recorded.

Table 1. ECS score for barrier assessment

Results

The ECS score moderately correlated with endoscopic scores (r=0.48 for MES, r=0.66 for UCEIS and r=0.63 for PICaSSO) and strongly with histological scores (r=0.69 for RHI, r=0.71 for NHI and r=0.84 for PHRI). Seven patients experienced adverse outcomes, with an ECS score <5 in the colon and <3 in the ileum predicting them (HR 2.35; p<0.01 and HR 5.78; p=0.05, respectively). All TJs were less expressed in UC patients than controls, with Claudin-2 and Occludin predominantly expressed in the epithelium and JAM-A mainly in the lamina propria. Notably, Claudin-2 cell density predicted outcomes (3370 vs 1290; p=0.01) and the ECS score in both colon and ileum correlated with Claudin-2 mean expression and cell density (ρ=.15; p=0.02 and ρ=.48, p<0.01, respectively). The combined intestinal barrier assessment by ECS and Claudin-2 cell density and mean expression significantly predicted outcomes in both colon and ileum (log rank test p<0.01).

Conclusion

ECS demonstrated promising ability to assess intestinal barrier in UC, positively correlating with TJ expression. Moreover, the novel barrier assessment through ECS complemented automated multispectral imaging in predicting adverse outcomes. These innovative tools hold promise in clinical practice for assessing barrier healing and guiding therapeutic management in UC and PSC colitis.

References

¹ Han IS, Baek DH, Hong SM, et al. Incidence and adverse clinical events of primary sclerosing cholangitis with ulcerative colitis. Int J Colorectal Dis. 2023;38:175. doi:10.1007/s00384-023-04464-z

Introduction

Patients with inflammatory bowel disease (IBD) in clinical and endoscopic remission may still experience disease relapse. Therefore, there is an unmet need for reliable predictors of outcomes to enable a personalized therapeutic approach. Recently, a novel histological score has highlighted the pivotal role of neutrophils in predicting disease outcomes in Ulcerative Colitis (UC)¹. Furthermore, the impairment of the intestinal barrier plays a key role in forecasting disease outcomes in UC and Crohn’s disease (CD). Hence, this study aims to assess the predictive role of neutrophils according to their tissue localization and intestinal barrier protein expression in IBD patients.

Aims & Methods

IBD patients in clinical remission who underwent colonoscopy between January 2020 and June 2022 at two tertiary referral centres were enrolled. Patients with a Mayo Endoscopic Score ≤1 for UC and a Simple endoscopic score of ≤6 for CD were included. The histological activity was assessed using the Robarts Histopathology Index, Nancy Histological Index, and PICaSSO Histological Remission Index. Neutrophil localization in the epithelium and lamina propria was carefully evaluated by experienced pathologists. Additionally, the immunohistochemical expression of barrier proteins, such as Claudin-2 and JAM (junctional adhesion molecule)-A, was assessed to evaluate the integrity of the intestinal barrier.

Results

Out of 60 UC and 76 CD patients, 60% were in histological activity. 25.8% of patients developed an adverse outcome within 12 months. Only neutrophils in the epithelium and not in lamina propria could predict adverse outcomes for UC (HR 5.20, p=.01) and CD (HR 4.38, p=.03) patients in endoscopic remission. Moreover, epithelial Claudin-2 expression correlated with endoscopic and histological disease activity and predicted outcomes in UC. Combining neutrophils in the epithelium or Claudin-2 expression significantly predicted outcomes in UC patients (HR 3.23, p=.035). Similar results were found for JAM-A in CD despite this protein showing less specificity as a barrier predictor of outcome.

Conclusion

This multicentre study highlights the potential role of epithelial neutrophil localization and Claudin-2 ‘leaky gut’ protein expression as practical clinical tools for predicting outcomes in IBD and potentially guiding patient-tailored therapy.

References

¹Gui X et al., Gut. 2022;71(5):889-898.

Disclosure

None

Introduction

Diagnosing and assessing Inflammatory Bowel Disease (IBD) requires invasive procedures such as endoscopy and intestinal biopsies. However, reliable and accurate non-invasive tools for assessing and monitoring inflammation in IBD patients are in an exciting phase of early development. Raman spectroscopy (RS), which provides molecular vibrational spectra, can characterise human biofluids and intestinal samples, offering the potential for non-invasive molecular assessment, characterisation and monitoring in IBD.

Aims & Methods

This study aims to explore the potential of RS applied to saliva, blood and intestinal biopsies from IBD patients for molecular characterisation and development of new biomarkers enabling a reliable and accurate non-invasive diagnosis of IBD, phenotyping between Crohn’s disease (CD) and Ulcerative Colitis (UC) and predicting mucosal healing (MH). IBD patients undergoing advanced endoscopy, i.e. ultra-high magnification endocytoscopy or probe-based confocal laser endomicroscopy, for disease assessment or surveillance and healthy controls were prospectively enrolled from two university hospitals in Cork, Ireland. Peripheral blood, saliva, and biopsies from the most representative bowel areas were collected. All matching samples were processed through a novel waveguide-enhanced RS technique, enabling sensitive detection of metabolites and other biochemical components. Additionally, MH from matching biopsies was assessed as RHI≤3 without lamina propria or epithelial neutrophils, NHI ≤1 and PHRI=0. RS data processing was performed using Matlab®. Spectral markers matched for saliva, blood and biopsies and differentiating IBD vs controls, CD vs UC and MH vs active inflammation were identified. A machine learning model based on linear discrimination analysis was used to assess the classification accuracy.

Results

A cohort of 16 IBD patients (median age 39, 11:5 M: F), comprising 9 UC and 7 CD, and 4 healthy controls (median age 43.5, 3:1 M: F) was enrolled, with matching saliva, blood, and biopsies. MH was found in 4 IBD patients. When comparing IBD patients with controls, differences in 3 wavenumbers were observed in all samples, corresponding to decreased tyrosine and lipids signals and increased carotenoids signal in IBD. Similarly, differences in spectral markers were found in CD vs UC, with increased lipids signal in CD and increased signals for carotenoids, nucleic acid, and amide proteins in UC. Interestingly, 5 matching differences in saliva, plasma and biopsies spectra were found in patients with MH compared to those with active inflammation, including an increase in nucleic acids and amide proteins and a decrease in phosphate, lipids, and carotenoids. The machine learning model showed promising accuracy (above 82%) of RS-aided biofluids analysis in diagnosing IBD and predicting phenotype and MH.

Conclusion

The combination of spectral markers from saliva and plasma, confirmed at tissue level, showed promise in accurately diagnosing IBD and distinguishing CD from UC. Notably, RS non-invasive biomarkers could predict MH with promising accuracy, identifying metabolites involved in IBD-associated inflammation, particularly lipids, carotenoids, and nucleic acids. Therefore, RS holds potential as a monitoring and predictive tool for inflammation and healing in IBD, with intriguing translational implications. Further studies are required to validate these preliminary findings and shed light on molecular mechanisms.

Introduction

Intestinal barrier integrity is emerging as a key therapeutic target in inflammatory bowel disease (IBD). Advanced endoscopic imaging techniques, including ultra-high magnification endocytoscopy (ECS) and probe-based confocal laser endomicroscopy (pCLE), enable real-time, in vivo structural and functional intestinal barrier assessment. Raman Spectroscopy (RS) can add a molecular dimension through vibrational fingerprinting of barrier-associated metabolites in biological samples.¹

Aims & Methods

This study explores the potential of RS integration with advanced endoscopy to characterise the intestinal barriers in IBD, targeting on tryptophan-derived metabolites and carbohydrates. A total of 46 IBD patients [25 Crohn’s disease (CD) and 21 ulcerative colitis (UC)] and 7 healthy controls (HC) undergoing endoscopic assessment or surveillance were prospectively enrolled from two academic centres. ECS or pCLE were used to evaluate epithelial and vascular barrier integrity. Barrier healing was defined as ECS score ≤ 1 or pCLE score ≤ 4, with preserved villi/crypt architecture, normal vasculature, and also absence of fluorescein leakage for pCLE. Advanced endoscopy-guided biopsies were collected from representative bowel segments. Mucosal healing (MH) was assessed by experienced pathologists using Nancy’s Histological Index ≤1. Epithelial (Claudin-2 and ZO-1) and vascular (PV-1 and CD-31) barrier markers were analysed through confocal microscopy, and their expression was automatically quantified using QuPath. Micro-RS was applied to tissue samples and matched sera to measure key tryptophan metabolites (kynurenine, indolepropionic acid, and melatonin) and carbohydrates.^2,3 Six-month major adverse outcomes (MAOs) were recorded. Group differences in Raman spectra were analysed with the Mann-Whitney test; correlations were assessed via Pearson’s test.

Results

Among patients undergoing ECS, barrier healing was observed in 10/22 (45%) colonic and 6/9 (67%) ileal cases. pCLE-based assessment revealed healing features in 5/16 (31%) colonic and 2/15 (13%) ileal evaluations. Raman spectral analysis showed a significant (p<0.05) reduction in tryptophan metabolites and carbohydrate-associated peaks in IBD tissues and sera compared to HC, especially in inflamed patients. When stratified by advanced endoscopy-identified barrier integrity, tryptophan metabolites -known barrier modulators-, and carbohydrates -likely reflecting mucin barrier layer- were markedly reduced (p<0.05) in patients with impaired barrier function. Notably, tryptophan derivatives positively correlated with Claudin-2 and ZO-1 in UC (r>0.4), and with PV-1 in CD (r>0.5). Similarly, carbohydrates correlated positively with Claudin-2 in UC (r=0.4) and negatively with CD-31 in CD (r=-0.4). Lower levels of indolepropionic acid and carbohydrates correlated with worse short-term MAOs (r<-0.4).

Conclusion

The integration of RS with advanced endoscopic images offers a novel, multi-layered approach to assess intestinal barrier integrity in IBD. RS can identify novel non-invasive biomarkers of mucosal and barrier healing, with potential real time applications for monitoring disease activity and predicting therapeutic outcome. Tryptophan metabolites may serve as a predictive biomarker of barrier healing paving the way toward personalised, barrier-targeted therapies.

References

1 Smith SCL, Banbury C, Zardo D, et al. PLoS One 2021; 16: e0252210.
2 Michaudel C, Danne C, Agus A, et al. Gut 2023; 72: 1296–307.
3 Arras W, Breugelmans T, Oosterlinck B, et al. J Crohns Colitis 2025; 19.