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

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

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

Celiac disease (CD) is a complex multi-systemic autoimmune disease. The present study aims to determine the rate of mucosal recovery and predictors of persistent mucosal damage after GFD. There is still little evidence on the best method for assessing gluten-free diet (GFD) adherence and mucosal recovery during treatment.

Aims & Methods

The retrospective study included only adult patients (age≥18 years old), with biopsy-proven CD evaluated at a tertiary referral centre between 2016-2021.We performed a logistic regression analysis to identify factors associated with partial mucosal recovery (MR) after GFD. We included in the multivariate analysis parameters available at the time of CD diagnosis.

Results

A total of 102 patients were enrolled, two thirds were females, median age of 39 yrs. The initial biopsy analysis showed different stages of villous atrophy (VA) in 79 (77.4%) cases, while 23(22.5%) cases showed mild enteropathy (Marsh 1,2). After at least 12 months of GFD, 26 (25.5%) patients had persistent VA despite good or excellent adherence to GFD. Younger patients (< 35yrs), who showed severe mucosal damage (Marsh 3c lesions) and who had increased anti-gliadin antibody (AGA) levels were at risk for failure to obtain mucosal recovery (MR). The multivariate Cox regression analysis demonstrated that complete mucosal atrophy (p = 0.007) and high AGA antibody levels (p = 0.001) were independent risk factors for a lack of mucosal improvement after at least 12 months of GFD. Interestingly, genotype, tTG-IgA antibody levels, or duration of GFD levels did not influence the occurrence of MR.

Conclusion

Although AGA seropositivity has lost much of their diagnostic significance in recent years due to the introduction of the more sensitive and specific antibody tests, our study reported that patients aged < 35yrs, who showed severe mucosal damage (Marsh 3c lesions) and who had increased AGA antibody levels at diagnosis were at risk for failure to obtain MR. The elevated AGA levels at diagnosis could be used as a prognostic tool for assessing MR.

Disclosure

nothing to disclose

Introduction

Glucose breath test (GBT) is the most commonly used method for the diagnosis of small intestinal bacterial overgrowth (SIBO). SIBO is a common condition with very unspecific symptoms (bloating, abdominal pain, diarrhea or constipation). Recently, American and European recommandations have been published to standardize the indications and the conditions to perform these tests.^1,2 However, some discrepancies remain between the European and American recommandations, and many statements were supported by low quality of evidence.^1,2 Moreover, several counfounding factors related to the patient’s condition may impact the result of the test, leading to a difficult interpretation.

Aims & Methods

This study aimed to assess the factors that could impact the results of a GBT. Therefore, we retrospectively reviewed the results of the GBT performed in our physiology department from 2013 to 2023. The test was performed after ingestion of 75g of glucose, with breath samples collected every 15min during 2 hours. The result was considered positive for hydrogen (H₂) if a rise ≥ 20ppm was observed or positive for methane (CH4) if a rise ≥ 10ppm was observed. Demographic data were collected, such as symptoms and questionnaires during the exam including Rome questionnaire, and the Gastro-Intestinal Quality of Life Index (GIQLI). Severity of symptoms was based on the GIQLI questionnaire, where a higher score represents a better condition. Medical and surgery history, such as drug treatments were then retrospectively collected from medical records, using a computerized research.

Results

A total of 2373 tests from different patients were included. The mean age was 46.0 ± 15.8 years old, with 1727 females (73.7%). The number of positive GBT was 576 tests (24.3%), with 294 tests positive for H₂ only, 214 tests positive for CH₄ only, and 69 tests positive for both. In univariate analysis, a higher age, female sex, and a lower body mass index (BMI) were associated with a positive GBT (Table). A history of diabetes, and a previous gastric surgery (sleeve, or bypass) were associated with a positive test, while proton pump inhibitors (PPI) or opioids intake did not change the outcome (Table). Regarding the symptoms, more severe abdominal bloating and constipation were associated with a positive test (1.28 ± 1.0 vs 1.41 ± 1.1; p=0.02, and 2.67 ± 1.3 vs 2.82 ± 1.2; p=0.03), while abdominal pain and diarrhea were not statistically different (1.57 ± 1.1 vs 1.67 ± 1.1; p=0.07; and 2.23 ± 1.3 vs 2.33 vs 1.4; p=0.14). In multivariate analysis, age (Odds Ratio (OR) = 1.02 [1.01 – 1.03]; p<0.001), BMI (OR=0.93 [0.90 – 0.96]; p<0.001), a previous gastric surgery (OR=9.32 [3.74 – 24.86]; p<0.001) and more severe constipation (OR=0.88 [0.79 – 0.98]; p=0.02) were associated with a positive GBT.

	Negative GBT (n=1797)	Positive GBT (n=576)	p value
Age (years)	44.7 ± 15.5	50.0 ± 16.2	<0.001
Sex (female)	1280 (71.2%)	447 (77.6%)	0.005
BMI (kg/m²)	25.0 ± 5.5	23.9 ± 5.2	<0.001
Diabetes	62 (3.5%)	30 (5.2%)	0.02
Gastric sleeve	8 (0.4%)	14 (2.4%)	<0.001
PPI intake	361 (20.1%)	123 (21.4%)	0.50
Opioids intake	183 (10.2%)	57 (9.9%)	0.91
Irritable bowel syndrome	561 (31.2%)	148 (25.7%)	0.09
Functional dyspepsia	704 (39.2%)	190 (33.0%)	0.08

Conclusion

The result of GBT is more likely to be positive in patients with constipation. A previous gastric surgery is a cofounding factor, leading to a non-interpretable result of the test. A diagnosis of irritable bowel syndrome or functional dyspepsia did not change the result of the test, such as PPI or opioid intake.

References

1. Hammer HF, Fox MR, Keller J, et al. European guideline on indications, performance, and clinical impact of hydrogen and methane breath tests in adult and pediatric patients: European Association for Gastroenterology, Endoscopy and Nutrition, European Society of Neurogastroenterology and Motility, and European Society for Paediatric Gastroenterology Hepatology and Nutrition consensus. United European Gastroenterol J 2022;10:15-40.
2. Pimentel M, Saad RJ, Long MD, et al. ACG Clinical Guideline: Small Intestinal Bacterial Overgrowth. Am J Gastroenterol 2020;115:165-178.