This study investigates how the immune system of children with celiac disease responds to controlled, small amounts of gluten. Children on a strict gluten-free diet are randomly assigned to receive either placebo, 50 mg of gluten, or 5 g of gluten once daily for three days, simulating real-life accidental exposure or dietary transgression. Blood samples are collected on Day 1 (before gluten intake) and Day 8 (five days after the last dose). Stool and urine samples are also collected for complementary analyses.
Using single-cell ribonucleic acid (RNA) sequencing, T-cell receptor sequencing, microRNA profiling, and exploratory metabolomics, the study aims to characterize changes in immune cell populations and gene expression after gluten exposure. The objective is to determine whether even very small amounts of gluten induce measurable systemic immune responses and whether these responses differ according to the dose administered. Understanding these mechanisms may support the development of new biomarkers and improve clinical management of pediatric celiac disease.
Age 8 to 14 years at study entry.
Diagnosis of celiac disease according to ESPGHAN 2020 criteria.
At least 18 months on a strict gluten-free diet (GFD).
Adequate adherence to the GFD, demonstrated by negative fecal gluten immunogenic peptides (GIP) prior to inclusion.
Obesity defined as BMI ≥ 95th percentile according to WHO criteria.
Diagnosed inflammatory bowel disease or diabetes mellitus.
Acute infectious illness at the time of inclusion.
Chronic hepatic, pulmonary, renal, or rheumatologic disease.
Parallel
Placebo
Dietary supplementPlacebo preparation identical in appearance and administration schedule to gluten doses. Administered once daily on Days 1, 2, and 3. Used as the comparator. Participants and evaluators remain blinded to allocation.
Gluten 50 mg
Dietary supplementParticipants receive 50 mg of gluten once daily on Days 1, 2, and 3. This dose simulates accidental low-level gluten exposure in children with celiac disease. Participants and evaluators remain blinded to allocation.
Gluten 5 g
Dietary supplementParticipants receive 5 g of gluten once daily on Days 1, 2, and 3. This dose simulates a dietary transgression in children with celiac disease. Participants and evaluators remain blinded to allocation.
Collection of blood, stool and urine samples on Day 1 and Day 8.
Procedure/SurgeryIn addition to blood samples, stool and urine samples will be collected for complementary analyses (including fecal gluten immunogenic peptides (GIP) and exploratory metabolomic assays)
Change in peripheral blood immune cell gene expression after controlled gluten ingestion
Change from baseline (Day 1) to post-intervention (Day 8) in gene expression profiles of peripheral blood immune cells measured by single-cell RNA sequencing (scRNA-seq).
Change in peripheral blood T-cell receptor repertoire after controlled gluten ingestion
Change from baseline (Day 1) to post-intervention (Day 8) in T-cell receptor (TCR) diversity and clonality in peripheral blood T cells measured by TCR sequencing
Dose-dependent changes in immune gene expression after gluten exposure
Change from baseline (Day 1) to post-intervention (Day 8) in immune-related gene expression levels measured by single-cell RNA sequencing (scRNA-seq), comparing placebo, 50 mg gluten, and 5 g gluten groups.
Dose-dependent changes in T-cell receptor repertoire clonality after gluten exposure
Change from baseline (Day 1) to post-intervention (Day 8) in T-cell receptor (TCR) clonality and diversity measured by TCR sequencing, comparing placebo, 50 mg gluten, and 5 g gluten groups.
Inter-individual variability in immune gene expression response to gluten exposure across participants
Inter-individual variability in changes from baseline (Day 1) to post-intervention (Day 8) in immune gene expression profiles measured by single-cell RNA sequencing (scRNA-seq).
Changes in serum exosomal microRNA expression after gluten exposure
Change from baseline (Day 1) to post-intervention (Day 8) in serum exosomal microRNA expression levels.
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Fundación Pública Andaluza para la Investigación de Málaga en Biomedicina y Salud
Lead sponsor
Hospital Regional de Malaga
Collaborator
Andaluz Health Service
Collaborator
Fundación Pública Andaluza para la Investigación Biomédica Andalucía Oriental
Collaborator
Universidad de Málaga
Collaborator
Consorcio Centro de Investigación Biomédica en Red (CIBER)
Collaborator
Biohealth Research Institute in Granada (ibs.GRANADA)
Collaborator
