Introduction

Clinical trials in coeliac disease require reliable histological metrics to quantify mucosal damage and demonstrate improvement with treatment. Regulatory agencies are recognizing this and expect some form of histological assessment in coeliac disease trials. Qualitative scoring systems are highly variable among readers. It has been suggested that quantitative scoring provides better consistency among different readers.

Aims & Methods

This study aimed to assess the variability that occurs when quantifying villus height (Vh), crypt depth (Cd) and the Vh:Cd ratio, and identified approaches to improve correlation among observers at different institutions. A set of 236 slides stained with haematoxylin and eosin from 28 patients with coeliac disease on a gluten-free diet (NCT03738475) were used. Quantitative scoring of Vh, Cd, Vh:Cd ratio and the sum of Vh and Cd were initially compared between different institutions that prioritized ocular microscopy or digital pathology. Additionally, the change from baseline with gluten ingestion for each of these measurements was compared between institutions. Improvements to inter-reader correlations were assessed by implementing a common digital pathology software and using agreed upon criteria for making quantitative measurements. Data sets were subjected to statistical analysis to understand correlation and significance. Regression slopes were calculated and the coefficient of determination, R², was derived as a measure of linearity. The concordance correlation coefficient was calculated as an index of agreement beyond linearity. Pairwise Pearson correlation coefficients between readers from different institutions were calculated to demonstrate the degree of association between readers.

Results

Initial inter-reader analysis found that Vh:Cd ratios had a similar, linear trend between readers, regardless of ocular or digital pathology usage, but that there was a consistent systematic difference partially due to differences in reader assessment of the location of the crypt–villus border. Use of a common digital pathology platform and clear scoring criteria greatly improved inter-reader correlations, even when scoring was done by a non-pathologist. Importantly, the assessment of change from baseline in the quantitative measurements after gluten exposure was also highly concordant (Table). The software also enabled visual comparison of readers’ individual measurements of the crypt and villous lengths allowing potential reasons for differences in these measurements to be identified.

Table 1. Changes from baseline in Vh and Cd metrics obtained via ocular microscopy and digital pathology after gluten challenge.

Metric	Substudy 1		Substudy 2	Difference from institution 2
	Digital pathology (institution 2)	Ocular microscopy (institution 1)	Digital pathology + training (institution 1)	Substudy 1 (institution 1)	Substudy 2 (institution 1)
Vh, μm	−95.25 (−130.84, −59.66)**	−38.01 (−72.90, −3.12)*	−87.3 (−137.5, −37.0)†	−57.24**	−7.95†
Cd, μm	50.61 (35.87, 65.34)**	7.73 (−0.26, 15.72)†	40.7 (16.6, 64.8)†	42.88**	9.91†
Vh:Cd ratio	−0.79 (−1.03, −0.54)**	−0.38 (−0.61, −0.15)*	−0.7 (−1.0, −0.4)†	−0.41**	−0.09†
Vh+Cd, μm	−44.64 (−74.62, −14.67)*	−30.28 (−67.44, 6.88)†	−46.5 (−83.9, −9.1)†	−14.36†	1.90†
*p < 0.05 for difference between digital and ocular reads. **p < 0.001 for difference from baseline (0). †p values were not significant (> 0.05). Data are mean (95% CI). Vh, Cd, Vh:Cd ratio and Vh+Cd measurements were obtained by ocular microscopy by one reader from institution 1 and by digital pathology by three readers from institution 2. Shown here are the changes in Vh and Cd metrics between baseline and Day 14 after gluten challenge and the changes (improvement) in the performance of the reader (same reader in each substudy) between substudies 1 and 2. Cd, crypt depth; CI, confidence interval; NR, not reported; Vh, villus height; Vh+Cd, sum of the villus height and crypt depth; Vh:Cd, villus height:crypt depth.

Conclusion

The results suggest that quantitative histology in coeliac disease is reliable and can be improved through digital pathology, software measurement aids and attention to selecting optimally oriented sections. Given the ease with which pathologists without training in the use of quantitative histology software can produce results similar to those from experienced central laboratories, quantitative histology may be feasible outside the clinical trial setting.

Disclosure

AV has nothing to disclose. DAL, DR, DC, GS, JM and QL are employees of Takeda Development Center Americas, Inc. and receive stock or stock options. JI is the founder and owner of Jilab, Inc.; is a Full Professor at Tampere University, Medical Faculty; and has a consulting agreement with Takeda. JAM has grants from Nexpep/ImmusanT, National Institutes of Health, Immunogenix, Takeda Pharmaceutical, Allakos, Oberkotter, Cour; and has received consultancy fees from Bionix, Lilly Research Laboratory, Johnson & Johnson, Dr. Schar USA, UCB Biopharma, Celimmune, Intrexon Corporation, Dren Bio, Reistone Pharma, Chugai Pharma, Kanyos, Boehringer Ingelheim, Equillium and Torax Medical. MR is a consultant for Takeda and Bristol Myers Squibb. MZC has nothing to disclose. TTW has nothing to disclose.
Funding statement: This study was sponsored by Takeda Development Center Americas, Inc. Medical writing support was provided by Oxford PharmaGenesis, Oxford, UK and was funded by Takeda Pharmaceuticals.

Introduction

Diagnostics of enteropathy with membrane digestion disturbance (EMDD) is based on the evaluation of carbohydrase activity in duodenal mucosa (DM). There is no information about diagnostic value of ileum mucosa (IM) carbohydrases.
Study objective. Assess the diagnostic value of carbohydrases activity in mucous membrane of duodenum and distal ileum in patients with EMDD and irritable bowel syndrome (IBS).

Aims & Methods

The study includes 12 patients with EMDD, 16 patients with IBS and 12 relatively healthy persons (control group). Study design included analysis of food tolerance and evaluation of activity of sucrase (S), glucoamylase (GA), maltase (М) and lactase (L) in mucous membrane of duodenum and ileum based on the method of Dahlquist with N.I.Belostotsky modification. Microsoft Office Excel and STATISTICA 10.0 were used for statistical evaluation of material. Mann-Whitney criterion was used to evaluate the differences between quantitative parameters. The differences were considered to be significant at p <0.05. Control group was used for comparison.

Results

Ratio of carbohydrase activity in duodenum and ileum in control group and patients with IBS and EMDD.

In control group the carbohydrases activity in duodenum mucosa was not significantly statistically different from the one in ileum mucosa.
In patients with IBS glucoamylase and maltase activity parameters in IM were significantly different from activity in DM, which can be interpreted as functional inequality of enzyme activity of various departments of small intestine.
In patients with EMDD GA level in ileum mucosa is 5.2 times higher, maltase level is 3.8 times higher and sucrose level is 2 times higher than duodenum enzyme levels, which demonstrates the possibility of formation of compensatory reaction of adaptive shift type towards distal segments of small intestine, initially described by A.M. Ugolev et al. in 1985

Conclusion

Enzyme activity distribution along small intestine is an adaptor function of its segments. Our study confirmed the need and promising outlook of further investigations in terms of evaluation of proximo-distal gradients of enzyme activity to study compensatory reactions which can be fulfilled by distal segment of small intestine in case of duodenum damage.
The evaluation of carbohydrase activity in ileum mucosa provides additional opportunities for diagnostics of EMDD and its differentiation from IBS, which can be used to improve therapeutic approaches.

Introduction

Celiac disease is an autoimmune disorder where the intake of gluten (a protein found in wheat, barley, and rye) causes damage to the small intestine and impairs nutrient absorption. Studies have shown that individuals with celiac disease have reduced quality of life and higher levels of anxiety and depression. However, this has not been studied in the general population, among individuals with newly discovered, untreated celiac disease.

Aims & Methods

The aim of this study was to investigate anxiety, depression, mental health, and subjective well-being in individuals with newly diagnosed and untreated celiac disease, compared with the general population. This study used data from the fourth Trøndelag health study (HUNT4), conducted between 2017 and 2019 with over 56,000 adult participants from 20 years of age. Previously undiagnosed celiac disease was identified among the participants by analyses of blood samples from all participants and confirmed with endoscopy and duodenal biopsies. The participants in HUNT4 also answered questionnaires measuring levels of anxiety and depression using the Hospital Anxiety and Depression Scale (HADS), mental distress using the Cohort of Norway Medical Health Index (Conor-MHI), and subjective well-being using a three-part questionnaire. Differences in mental health among individuals with newly diagnosed celiac disease and the remaining HUNT4 participants were compared.

Results

Celiac disease was diagnosed in 454 HUNT4 participants by March 2022. The celiac group had a lower median age (53 vs. 56 years) compared to the remaining HUNT4 population. The celiac group had significantly lower mean scores on the HADS anxiety subscale (4.0 vs. 4.4), depression subscale (2.8 vs. 3.3), and total scale (6.8 vs. 7.7) compared to the reference population. On the CONOR-MHI scale, the celiac disease group had a lower total mean score (1.4 vs. 1.5) with lower scores in the categories of "secure and calm", "irritable", "happy and optimistic", "sad/depressed", and "lonely" compared to the reference population. Finally, the celiac disease group had a lower mean score on the question “Do you feel mostly strong and clear, or tired and fatigued?” (3.3 vs. 3.4) and a higher mean score on the question “How is your health now?” (3.0 vs. 2.9) compared to the reference population. No significant difference was observed in the HADS or CONOR-MHI scores by Marsh grades at diagnosis among the celiac disease group.

Conclusion

The study suggests that individuals with undiagnosed celiac disease have lower levels of anxiety, depression, and mental distress compared to the general population, while also reporting slightly better subjective well-being. These findings contradict other studies on psychological health in celiac disease.

Disclosure

No con

Introduction

Fatigue, defined as “an overwhelming sense of tiredness, lack of energy and feeling of exhaustion” [1] is a frequent complaint in celiac disease. A strict, lifelong gluten free diet is the only established treatment for celiac disease, however its long-term effect on fatigue is unknown.

Aims & Methods

Our aims were to investigate the course of fatigue severity and prevalence in celiac patients from diagnosis to five to seven years on a gluten free diet, and to investigate whether diet adherence, disease activity and selected biochemical markers are associated with fatigue.
We conducted a single-center prospective study in 45 patients with newly diagnosed celiac disease. The primary endpoints were fatigue severity, as measured by the Fatigue Severity Scale (FSS) [2], the fatigue Visual Analog Scale (fVAS) [3], and the Vitality subscale of the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36vs) [4]. SF-36vs scores were inverted (100 – SF-36vs = inverted SF-36vs) to ease comparison between the other scales, so that higher scores indicate more fatigue. Clinically relevant fatigue was defined by an FSS score of ≥4, a fVAS score of ≥50, and an inverted SF-36vs score of ≥65. Study visits were performed at the time of diagnosis (Visit 1), after adhering to a gluten free diet for one year (Visit 2) and for five to seven years (Visit 3). Diet adherence was evaluated through urinary gluten immunogenic peptides (GIP) and the Celiac Diet Adherence test (CDAT) [5]. Disease activity was assessed through Celiac Symptom Index (CSI) [6], celiac autoantibodies (anti tissue transglutaminase IgA and deamidated gliadin peptide IgG) and histologic evaluation (Marsh score). Nutritional status was assessed through BMI, hemoglobin, 25-hydroxy vitamin D, cobalamin, ferritin and folic acid levels.

Results

Fatigue severity was significantly reduced from time of diagnosis, for all three instruments (Table 1). Fatigue prevalence rates were significantly decreased from Visit 1 (40-53%) to Visit 3 (27-31%). Fatigue was significantly lower in patients with good adherence compared to patients with poor adherence according to CDAT scores (For FSS p=0.022, fVAS p=0.017, and for inverted SF-36vs p=0.033). There was however no difference in fatigue severity between adherent and nonadherent patients when evaluating adherence through urinary GIP. Fatigue was lower in patients in clinical remission according to CSI scores (for FSS p=0.002, fVAS p=0.004, and for inverted SF-36vs p=0.002), but not associated with autoantibodies or nutritional status. In multiple linear regression analysis, depression was the only independent variable significantly associated with all three fatigue measures at Visit 3, whereas pain was positively associated with inverted SF-36vs scores.

Table 1.

Fatigue instrument	Visit 1	Visit 2	Visit 3	Change from Visit 1 to Visit 3 (95% CI)	P-value Visit 1 vs. Visit 3	P-values across three time points (Visit 1, 2 and 3)
FSS	3.7 (2.2-5.0)	1.9 (1.5 - 3.3)	2.3 (1.3 - 4.2)	-0.5 (-1.2 – -0.2	0.012	<0.001
fVAS	45 (16.5-68.5)	15 (9.0 - 41.0)	21 (4.0 - 56.0)	-10 (-23.0 – -3.0)	0.003	0.001
SF-36vs (inverted)	65.0 (40.0-77.5)	30.0 (25.0 - 47.5)	45.0 (30.0 - 70.0)	-5.0 (-10 – 0)	0.006	<0.001

Conclusion

Fatigue remains a frequent complaint in celiac disease even after five to seven years on a gluten free diet. However, fatigue severity is reduced from time of diagnosis. Fatigue was associated with dietary adherence as assessed by CDAT. Disease activity according to CSI was associated with fatigue, whereas nutritional status, autoantibodies and histologic changes were not.

References

1. Krupp, L.B. and D.A. Pollina, Mechanisms and management of fatigue in progressive neurological disorders. Curr Opin Neurol, 1996. 9(6): p. 456-60.
2. Krupp, L.B., et al., The fatigue severity scale. Application to patients with multiple sclerosis and systemic lupus erythematosus. Arch Neurol, 1989. 46(10): p. 1121-3.
3. Wolfe, F., Michaud K, Pincus T, Preliminary evaluation of a visual analog function scale for use in rheumatoid arthritis. J Rheumatol, 2005. 32(7): p. 1261-6.
4. Ware, J.E., Jr. and C.D. Sherbourne, The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care, 1992. 30(6): p. 473-83.
5. Leffler, D.A., et al., A simple validated gluten-free diet adherence survey for adults with celiac disease. Clin Gastroenterol Hepatol, 2009. 7(5): p. 530-6, 536.e1-2.
6. Leffler, D.A., et al., A validated disease-specific symptom index for adults with celiac disease. Clin Gastroenterol Hepatol, 2009. 7(12): p. 1328-34, 1334.e1-3.

Disclosure

Berit Mære Skjellerudsveen: Received lecture honoraria from Takeda for lectures on IBD and pregnancy. This study is partly funded by a grant from the patient organization “The Norwegian Coeliac Society”.

Introduction

Intestinal ischemia-reperfusion injury (IRI) is a life-threatening condition caused by an abrupt obstruction of mesenteric blood flow. Reestablishing blood flow (reperfusion) is necessary to save intestinal tissue but unintentionally exacerbates damage induced by ischemia. Mitochondria are sensitive to IRI; if damaged, they release cytotoxic molecules that can aggravate injury. Therefore, removal of damaged mitochondria is essential for cellular survival. The selective clearance of mitochondria via autophagy, mitophagy, has been reported to protect against IRI in other organs, but its role in the intestine remains unknown.

Aims & Methods

The main objective of this study was to assess mitochondrial damage and evaluate signs of mitophagy in response to hypoxia-reoxygenation in a human small intestinal organoid (SIO) model and the differences therein between crypt-like (CL) and villus-like (VL) organoids. Both SIOs phenotypes were exposed to hypoxia-reoxygenation, and mitochondrial damage was assessed by live-cell imaging of mitochondrial membrane potential using fluorescent dye tetramethylrhodamine methyl ester (TMRM). In addition, ultrastructural changes in mitochondria were evaluated by transmission electron microscopy (TEM). Finally, mitochondrial content, autophagy, and mitophagy markers were assessed using quantitative PCR (qPCR) and Western blot.

Results

A drastic decrease in TMRM fluorescence, indicating impaired mitochondrial activity, was mainly observed in VL organoids and not in CL organoids. Mitochondrial ultrastructural changes were observed in both phenotypes, including mitochondrial swelling and disrupted cristae following hypoxia, whereas after 480 minutes reoxygenation, mitochondria exhibited a tubular or small round shape. Mitochondrial content markers were decreased in both organoid phenotypes following hypoxia-reoxygenation. Most importantly, only CL organoids showed a significant increase in autophagy and mitophagy markers.

Conclusion

Mitochondria of VL organoids are more severely damaged by hypoxia-reoxygenation, whereas CL organoids might be protected by inducing mitophagy. The occurrence of mitophagy is currently further validated with live-cell imaging.

Introduction

Infliximab (IFX) is a mainstay in the treatment of Crohn´s disease (CD) and ulcerative colitis (UC)^1,2. However, some patients do not respond to IFX; others develop antibodies to IFX (ATI), resulting in loss of response (LOR) over time^3,4. As second-line treatments for adults are not approved in pediatrics, current ECCO/ESPGHAN guidelines recommend combination therapy with an immunomodulator (IM) to improve response and prevent ATI development.^2,5 Recent studies suggested that combination therapy is not superior to IFX monotherapy if proactive therapeutic drug monitoring (TDM) is performed⁶.

Aims & Methods

We investigated whether recommended early combination therapy has an additional benefit in a proactively managed pediatric IBD cohort. Medical records from 01/2013 to 12/2022 were retrospectively reviewed and children (3-18 years) with at least four doses of IFX, 3-60 months (m) follow-up and proactive TDM were included. Patients were grouped in either early (IM^e: ≤3m since IFX start and duration of IM therapy >6m or until the end of observation) or late/never IM treatment (IM^l/n: >3m since IFX start/no IM combination therapy). Kaplan-Meier analysis and log-rank test (significance level p<0.05) were used to analyze time to LOR with IFX discontinuation or time to ATI development. Hazard ratio (HR) applying Cox regression represents the relative risk of LOR occurring in IM^l/n compared to IM^e.

Results

Of 315 reviewed IBD children, 127 patients were included (43% girls, median age at IFX start: 14 years, 77 CD, 50 UC including 5 IBD-u) and IFX treatment of 2855 visits was analysed. Sixty patients received IM^e (41 CD, 19 UC), 20 patients started IM >3m after IFX (13 CD, 7 UC) and 47 patients had IFX monotherapy (23 CD, 24 UC). Patient characteristics, previous therapies, IFX dosage and intervals were comparable between IM^e and IM^l/n groups (except E2 in UC). Proactive TDM was comparable in both IM^e and IM^l/n, i.e., duration from IFX start to first TL measurement (median of 6 weeks, p=0.9), median number of 4 TL measurements per follow-up year (p=0.6), and median TL of 10 and 11 μg/mL (p=0.6), respectively. Most patients (84%) had a median TL >5µg/ml over time. The median follow-up time was 29.6m for IM^e and 25.5m for IM^l/n.
IFX treatment persistence was 68% after 60m in the total cohort. LOR with IFX discontinuation was observed in 7 IM^e and 15 IM^l/n patients (p=0.2). Combining IFX infusions with IM^e significantly delayed the occurrence of LOR with IFX discontinuation (median LOR free interval IM^e 29.4m, IM^l/n 8.6m, p=0.01). The IM^l/n group was more likely to experience LOR than IM^e after 1, 2, 3, 4, and 5 years (HR₁=10.3, p₁=0.01; HR₂=4.1, p₂=0.02; HR₃=2.9, p₃=0.03; HR₄=2.9, p₄=0.03; HR₅=2.1, p₅=0.09). IM^l/n instead of IM^e increased the risk for LOR, especially in UC patients (HR 7.5, p=0.04).
Low ATI >10AU/ml were found in 76/106 IFX-treated patients, while high ATI >100AU/ml were detected in 10/52 IM^e and 15/54 IM^l/n patients, p=0.2. There was no significant difference regarding median ATI level (both 12AU/ml, p=0.7) and ATI-free interval (9m vs 8m, p=0.65) in the groups. Only in CD patients, IM^l/n showed a significantly higher risk of developing high ATI (HR 4.1, p=0.046) compared to IM^e.

Conclusion

Proactive TDM resulted in high IFX treatment persistence in pediatric IBD. Early combination therapy of IM with IFX showed a significant benefit with improved IFX treatment persistence during the first four years of therapy. This effect was partly explained by preventing high-titer ATI in CD patients.

References

1. van Rheenen, P. F. et al. The Medical Management of Paediatric Crohn’s Disease: an ECCO-ESPGHAN Guideline Update. J. Crohns Colitis 15, 171–194 (2021).
2. Turner, D. et al. Management of Paediatric Ulcerative Colitis, Part 1 and 2: Ambulatory Care-An Evidence-based Guideline from European Crohn’s and Colitis Organization and European Society of Paediatric Gastroenterology, Hepatology and Nutrition. J. Pediatr. Gastroenterol. Nutr. 67, 257–291 (2018).
3. Adedokun, O. J. et al. Pharmacokinetics of infliximab in children with moderate-to-severe ulcerative colitis: results from a randomized, multicenter, open-label, phase study. Inflamm. Bowel Dis. 19, 2753–2762 (2013).
4. Vande Casteele, N. et al. Antibody response to infliximab and its impact on pharmacokinetics can be transient. Am. J. Gastroenterol. 108, 962–971 (2013).
5. van Rheenen, P. F. et al. The Medical Management of Paediatric Crohn’s Disease: an ECCO-ESPGHAN Guideline Update. J Crohns Colitis (2020) doi:10.1093/ecco-jcc/jjaa161.
6. Sandborn, W. J. et al. Treating beyond symptoms with a view to improving patient outcomes in inflammatory bowel diseases. J. Crohns Colitis 8, 927–935 (2014).