Crosstalk Between Mucosal-Associated Invariant T Cells and the Gut Microbiota and Mucosa in the Development of Type 1 Diabetes in Children
试验速览
- 阶段
- 不适用
- 状态
- 招募中
- 发起方
- 入组人数
- 180
- 试验地点
- 3
- 主要终点
- blood MAIT cells frequency
研究概览
简要总结
To investigate in a prospective way changes in Mucosal-Associated Invariant T (MAIT) cells frequency, phenotype and function in link with the gut microbiota, gut integrity and the presence of Coxsackie virus B in two cohorts of pediatric patients: patients with a high genetic risk of type 1 diabetes and pediatric patients with recently diagnosed T1D by comparison with control subjects
Tasks:
- To measure blood MAIT cells frequency, phenotype and function in the three cohorts
- To analyze gut microbiota and the presence of Coxsackie B enterovirus (CVB) and their impact on MAIT cell function
- To evaluate gut integrity and analyze the gut mucosa
- To integrate all the data obtained with T1D development and evolution
详细描述
Subjects: Multi study. Subjects will be included in the pediatric diabetes and endocrinology unit in Necker Sick children hospital and in the pediatric unit of ANTONY hospital.
- Recent-onset (RO) n=40: New onset patients will be included shortly after diagnosis.
- At risk (AR) cohort n=70: Routinely screened siblings of T1D patients previously tested positive for HLA DR3 and DR4 will be asked to be a part of the study.
- Control (C) cohort (n=50): Control subjects will be patients consulting at Necker Hospital for endocrine testing
- Control for Endoscopy (CE) n= 20: patients consulting at Necker Hospital or at Antony hospital for UGI endoscopy
Analysis:
MAIT cell analysis: For FACS analysis, MAIT cells will be identified as CD3+ CD4- CD161high Vα7.2+ T cells. Surface markers will be analyzed to determine their activation status (CD25, CD69, CD44), their exhaustion (PD1, KLRG1, TIM3), their migration capacity (CCR6), and their proliferation and survival will be analyzed by Ki67 and BCL2 expression. Cytokine production will be assessed after PMA-ionomycin activation, followed by intracytoplasmic staining with antibodies against IL-2, IFN-γ TNF-α, IL-2, IL-4, IL-10, IL-13, IL-17 and granzyme B. To determine the capacity of MAIT cells to response to TCR stimulation (exhaustion), in vitro stimulation will be performed in the presence of specific bacterial ligands. Activation marker expression will be analyzed by FACS and cytokines released in the supernatant by Cytometry based assay.
Gut microbiota analysis: Stool samples are collected and directly kept under anaerobic condition. Within an hour the samples are processed: one fraction is aliquoted and frozen at -80°C and another fraction is used to prepared fecal supernatant for the bioassay of MAIT ligands, aliquoted and frozen. Bioassay to measure the presence of MAIT cell ligands by bioassays using WT3 cell line as well as plate bound MR117. 16S sequencing of all samples and according to the results obtained with the bioassay and 16S, 10 samples will be selected for metagenomic analysis.
研究设计
- 研究类型
- Observational
- 观察模型
- Case Control
- 时间视角
- Prospective
入排标准
- 年龄范围
- 12 Months 至 15 Years（Child）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •Recent onset group
- •age > 12 months and < 15 years
- •recently diagnosed type 1 diabetes according ISPAD criteria
- •At risk subjects:
- •age > 12 months and < 15 years
- •siblings of type 1 diabetic patient
- •HLA DR3 and DR4 positive
- •Control subjects:
- •age > 12 months and < 15 years
- •no HLA associated with high risk type 1 diabetes
- •no antibodies against pancreas antigenes
- •Control subjects for UGI endoscopy:
- •age > 12 months and < 15 years
- •suspicion of coeliac disease or gastritis
排除标准
- •For all groups:
- •no health care insurance
- •parents or tutors unable to sign the consent
- •personal history of autoimmune disease and/or inflammatory disease except from T1D for RD and CE groups
- •use of corticosteroids during the month before inclusion
- •pregnant subjects
- •medical contraindication of anesthetic topics
- •For control subjects for UGI endoscopy control and Recent onset-endoscopy group:
- •age below 8 years for Recent onset-endoscopy group
- •age below 4 for UGI endoscopy control group
- •cardiac or respiratory insufficiency, cardiac rhythm disorders, coagulation disease, patients treated with anticoagulant or antiaggregant drug
- •history of allergy to anesthetic drug
研究组 & 干预措施
recent onset patients
patients with recently diagnosed type 1 diabetes
干预措施: MAIT cells analysis (Diagnostic Test)
recent onset patients
patients with recently diagnosed type 1 diabetes
干预措施: MAIT cytokines production analysis (Diagnostic Test)
recent onset patients
patients with recently diagnosed type 1 diabetes
干预措施: Lactulose/Mannitol Test (Diagnostic Test)
recent onset patients
patients with recently diagnosed type 1 diabetes
干预措施: UGI Endoscopy (Procedure)
recent onset patients
patients with recently diagnosed type 1 diabetes
干预措施: Coxsackie virus B infection status (Diagnostic Test)
at risk patients
patients with a high genetic risk type 1 diabetes
干预措施: MAIT cells analysis (Diagnostic Test)
at risk patients
patients with a high genetic risk type 1 diabetes
干预措施: MAIT cytokines production analysis (Diagnostic Test)
at risk patients
patients with a high genetic risk type 1 diabetes
干预措施: Lactulose/Mannitol Test (Diagnostic Test)
at risk patients
patients with a high genetic risk type 1 diabetes
干预措施: Coxsackie virus B infection status (Diagnostic Test)
control subjects
control patients (no risk of type 1 diabetes and no diagnosed type 1 diabetes)
干预措施: Coxsackie virus B infection status (Diagnostic Test)
control subjects
control patients (no risk of type 1 diabetes and no diagnosed type 1 diabetes)
干预措施: MAIT cells analysis (Diagnostic Test)
control subjects
control patients (no risk of type 1 diabetes and no diagnosed type 1 diabetes)
干预措施: MAIT cytokines production analysis (Diagnostic Test)
control subjects
control patients (no risk of type 1 diabetes and no diagnosed type 1 diabetes)
干预措施: Lactulose/Mannitol Test (Diagnostic Test)
control subjects for endoscopy
patients without type 1 diabetes requiring UGI endoscopy for any medical reason
干预措施: MAIT cells analysis (Diagnostic Test)
control subjects for endoscopy
patients without type 1 diabetes requiring UGI endoscopy for any medical reason
干预措施: MAIT cytokines production analysis (Diagnostic Test)
control subjects for endoscopy
patients without type 1 diabetes requiring UGI endoscopy for any medical reason
干预措施: UGI Endoscopy (Procedure)
control subjects for endoscopy
patients without type 1 diabetes requiring UGI endoscopy for any medical reason
干预措施: Coxsackie virus B infection status (Diagnostic Test)
结局指标
主要结局
blood MAIT cells frequency
时间窗: Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group)
percentage of MAIT cells in the peripheral blood
次要结局
- MAIT cells membrane markers analysis(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- Cytokines production in the cytoplasm of the MAIT(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- Evaluation of gut mucosal integrity(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- Presence of CVB in the stools(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- MAIT cell ligands measurements(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- Blood immunoglobulins against CVB infection statu(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- Measurement of intestinal gut mucosal permeability(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))
- 16 S sequencing in the Subjects stools(Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group))