The Effect of Gluten-free Diet on Residual Beta-cell Capacity, Immune Function and Gut Microbiome in Children With Newly Diagnosed Type 1 Diabetes (T1D)
Phase: NA
Conditions: Type 1 Diabetes
Sponsor: University Hospital, Motol

The effect of gluten-free diet on type 1 diabetes (T1D) 
 
NCT02867436 
 
January 10th, 2016 
 
 

Trial design 
This study is a prospective non-randomized intervention trial.  
 
Participants 
Study participants were recruited from children patients diagnosed with T1D at inpatient 
pediatric diabetes clinics at two centers in Prague, Czech Republic (University Hospital Prague-
Motol and University Hospital Prague-Královské Vinohrady). Both centers are tertiary care 
hospitals that annually admit over 65 children patients with newly diagnosed T1D. The 
recruitment will last from January 2016 to January 2018. Subjects and their parents have the 
right to withdraw from the study at any time without giving the reason. 
 
Eligibility criteria 
Following inclusion criteria will be applied: 
• T1D diagnosed according to the ADA criteria [1] 
• age 4 – 17.99 years at onset 
• positivity of a least one T1D associated antibody (IAA, anti-IA2, anti GAD65) 
• presence of high risk HLA-DQB1*03:02-DQA1*03 or DQB1*02-DQA1*05 molecules 
• no coeliac disease and negativity of anti-tissue transglutaminase antibodies 
• body mass index below +2SD of the age and height standard 
• no concomitant disease potentially influencing immune response or gluten sensitivity 
 
Intervention 
Subjects in the intervention group will follow strict gluten-free diet while receiving the 
recommended daily doses of macronutrients. Their meal records for three days prior to the visit 
will be required for each visit as well as a consultation with nutrition specialist. Adherence to the 
diet will be tested via the negativity of the gluten immunogenic peptide (GIP) in the commercial 
iVylisa GIP-S (Biomedal, S.L., Sevilla, Spain) ELISA kit. Stool samples will be tested for GIP 
presence every 3 months without especially notifying the subjects. Subjects in the intervention 
arm will receive financial reimbursement for the added cost of gluten-free edibles.  
 
 

Study procedure 
The caregivers of the subjects will sign a written informed consent with the study protocol prior 
to the enrollment in the first 30 days after T1D diagnosis. 
All subjects will be hospitalized at T1D onset. During the hospitalization, a blood sample will be 
taken for the measurement of anti-GAD65, anti-IA2 and IAA, glycosylated hemoglobin (HbA1c) 
(performed by high liquid chromatography) and screening for coeliac disease (total IgA to rule 
out IgA deficiency anti anti-tissue transglutaminase antibodies analyzed by ELISA test). A stool 
sample will also be taken for further analysis of their gut microbiome content. 
If the subjects fulfill the inclusion criteria, they will be offered to join the trial. The subjects and 
their parents will choose their study group freely and a mixed-meal tolerance test (MMTT) will 
scheduled between 4-8 weeks after T1D onset. 
The MMTT is considered a gold standard for the measurement of the endogenous production of 
residual insulin in subjects with T1D [2]. MMTTs will be performed for all subjects at group 
allocation and at 6 and 12 months. All subjects will receive a standard liquid meal Ensure Plus 
(Abbott Laboratories BV, Zwolle, Netherlands) and their C-peptide levels will be measured at 
the baseline and each 30 minutes up to 150 minutes. All MMTTs will be performed after 
overnight fasting and children will receive only their evening dose of long-acting insulin 
analogue. The MMTT will be rescheduled if the subjects` capillary glucose is <3.9 mmol/L (<70 
mg/dL) or >10 mmol/L (>180 mg/dL). Serums of subjects from the second center will be 
transported at 4-8°C within 48 hours to the central laboratory. Immediately after the first MMTT, 
subjects who will choose the intervention group will start with strict gluten-free diet. After the 
third MMTT at 12 months, subjects and their parents will be allowed to discontinue the diet at 
their will.  
Subjects will be followed up at 3 months intervals. During each visit subjects will undergo a 
physical examination and their body weight, height and BMI will be measured. Their HbA1c 
will be measured and their total daily insulin dose noted.  
Stool samples will be taken at T1D onset, allocation visit and each 3 months up to 15 months for 
microbiome analysis and adherence test if the patient belongs to the intervention group. The 
stool samples will be kept at -80°C until the DNA extraction using the PowerSoil DNA isolation 
kit (Qiagen, Hilden, Germany). Microbiome profiles will be determined by next-generation 
sequencing of the V4 variable region of 16S rDNA and processed in a bioinformatic pipeline as 
described previously [3]. 
Blood samples for the measurement of regulatory cell subsets (measured by flow cytometry) and 
their cytokine profiles (after short term PMA-ionomycin re-stimulation) will be taken at baseline 
and at 6 and 12 months. 

Intestinal permeability will be measured through serum zonulin (a marker for dysfunctional gut 
barrier function) levels at baseline and at 12 months [4]. In addition, urine lactulose/mannitol 
ratio following the ingestion of lactulose/mannitol solution (mannitol g, lactulose g in 100ml 
water for injection) and 6 hours urine collection [5] will be performed at 12 months (measured 
using high-performance liquid chromatography). 
 
End points 
Primary 
• Area under the curve (AUC) of the C-peptide level during the mixed meal tolerance tests 
Secondary 
• HbA1c 
• Insulin requirement (IU/kg) 
• Insulin dose adjusted HbA1c (IDAA1c) 
• Gut microbiome composition 
• Immune parameters (regulatory cell subsets, cytokine production) 
• Intestinal permeability (zonulin concentration and lactulose/mannitol urine excretion 
ratio) 
 
Data collection nad management 
All participants will be assigned a study identification number. The forms with patient data will 
be stored in a locked room with access only to the team of researchers at both centers. Electronic 
version of data will be kept in password protected databases in accordance with the GDPR 
legislature. 
 
Statistical analysis 
The analysis of the primary outcome will be based on an ANCOVA model with C-peptide AUC 
at 12 months as a response and the intervention assignment, baseline C-peptide AUC at visit 1, 
age, gender and body weight as covariates.  The tests and confidence intervals will use robust 
sandwich variance estimator. The primary analysis will follow the per-protocol principle. 
Secondly, the results at 6 months will also be analyzed. Analyses of secondary outcomes 
(HbA1c, insulin dose, immune parameters and gut microbiome composition) will proceed by 
similar methodology. Longitudinal analyses will be performed by linear generalized estimating 
equation models with a working independence structure. Linear trends in continuous outcomes 

over 12 months will be estimated by the intervention arm and compared with adjustment for sex, 
age, and baseline body weight SDS. 
 
Ethics and dissemination 
The study follows the CONSORT guidlines [6] and was approved by the Ethics Commitee of the 
2nd Faculty of Medicine of the Charles University in Prague. The study protocol was registered 
at ClinicalTrials.gov (NCT 02867436). Written informed consent will be presented and signed 
by all the participants caregivers. All deviations from the protocol will be monitored and duly 
noted to assure a safe conduct. The results will be submitted to peer-reviewed journals and 
abstracts will be presented at national and international conferences. All investigators will have 
access to the final dataset. 
 
 

References 
 
1. 
American Diabetes, A., (2) Classification and diagnosis of diabetes. Diabetes Care, 2015. 38 
Suppl: p. S8-S16. 
2. 
Greenbaum, C.J., et al., Mixed-meal tolerance test versus glucagon stimulation test for the 
assessment of beta-cell function in therapeutic trials in type 1 diabetes. Diabetes Care, 2008. 
31(10): p. 1966-71. 
3. 
Cinek, O., et al., Imbalance of bacteriome profiles within the Finnish Diabetes Prediction and 
Prevention study: Parallel use of 16S profiling and virome sequencing in stool samples from 
children with islet autoimmunity and matched controls. Pediatr Diabetes, 2017. 18(7): p. 588-
598. 
4. 
Esnafoglu, E., et al., Increased Serum Zonulin Levels as an Intestinal Permeability Marker in 
Autistic Subjects. J Pediatr, 2017. 188: p. 240-244. 
5. 
Sequeira, I.R., et al., Standardising the lactulose mannitol test of gut permeability to minimise 
error and promote comparability. PLoS One, 2014. 9(6): p. e99256. 
6. 
Schulz, K.F., et al., CONSORT 2010 changes and testing blindness in RCTs. Lancet, 2010. 
375(9721): p. 1144-6.