Association of Breg and Treg With the Clinical Effects of Infliximab in the Treatment of Patients With Crohn's Disease
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 发起方
- 入组人数
- 32
- 试验地点
- 1
- 主要终点
- Change of the frequency of Breg in N group
研究概览
简要总结
Aims: The main aim of this study is to access the predictive value of Treg and Breg for the clinical effect of Infliximab in the treatment through analyzing the relationship between Breg and Treg and the efficacy of Infliximab.
Design: It is a prospective, observational study. In the treatment group, 32 patients with Crohn's disease (CD) about to start Infliximab-treatment are recruited. They have blood samples drawn at week 0 and 14 of Infliximab treatment. 33 healthy individuals serve as a control group. Controls are only investigated once. All treatment and follow-up are according to national guidelines. The frequencies of Treg and Breg are investigated using flow cytometry. Subjects data are extracted from various registries.
详细描述
- Patients with CD at initial active stage are collected from department of gastroenterology, the Second Affiliated Hospital of Wenzhou Medical University. The diagnosis of CD is based on clinical, endoscopic, laboratory, radiologic, histo-pathological findings and close follow-up in accordance with the guidelines for the diagnosis and treatment of CD issued by the European Crohn's Disease and Colitis in 2016. Healthy individuals are collected from the Health Examination Center of the Second Affiliated Hospital of Wenzhou Medical University as the healthy controls. The demographic and characteristic information of CD patients and healthy controls are recorded.
- Infliximab (5mg/kg) is given intravenously at week 0, 2 and 6 to induce CD remission, and then maintained with the same dose of Infliximab every 8 weeks.
- CD patients are followed for 14 weeks after the first administration of infliximab. At week 14 of Infliximab treatment, according to symptoms, CDAI and endoscopic mucosal healing, CD patients are classified as remission group (CDAI<150 and endoscopic mucosal healing, R group) and non-remission group (CDAI≥150 and/or mucosal non-healing group, N group).
- C-reactive protein (CRP), leucocyte count (WBC), platelet count (PLT), erythrocyte sedimentation rate (ESR) are detected in CD patients to assess the clinical efficacy at week 0 and 14 of Infliximab treatment respectively.
- Approximate 5 mL of peripheral fasting venous blood is obtained from every CD patient (at weeks 0 and 14 of Infliximab treatment) and healthy controls. Peripheral blood mononuclear cells (PBMCs) are isolated from the blood samples.
- Multi-color flow cytometry is applied to examine the frequency of Breg (CD3-CD19+IL-10+ B cell) in B cell: PBMCs are resuspended in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin Solution, added in a 96-well flat-bottom culture plate. Then the obtained cells are stimulated with lipopolysaccharide for 48 hours, cultured at 37℃ in 5% CO2 in the incubator, and added Phorbol 12-myristate 13-acetate and ionomycin and Brefeldin A during the final 5 hours. After stimulated and cultured in vitro, PBMCs are stained with FITC-conjugated anti-human CD3 antibodies, APC-conjugated anti-human CD19 antibodies for 30 minutes with blocking light at 4℃. Then stained cells are fixed and permeabilized using a Cytofix/Cytoperm kit and stained with PE-conjugated anti-human IL-10 antibodies for 2 hours at 4℃ that protected form light. Flow staining buffer solution resuspends cells before flow cytometry detection of Breg.
- Multi-color flow cytometry is applied to examine the frequency of Treg (CD4+CD25+Foxp3+ T cell) in CD4+ T cell: PBMCs are stained with FITC-conjugated anti-human CD4 antibodies, APC-conjugated anti-human CD25 antibodies for 30 minutes with blocking light at 4℃. Stained cells are added fixing buffer and incubated at 4℃ in the dark overnight, then stained with PE-conjugated anti-human FoxP3 antibodies for 2 hours at 4℃ that protected form light. Flow staining buffer solution resuspends cells before flow cytometry detection of Treg.
研究设计
- 研究类型
- Observational
- 观察模型
- Cohort
- 时间视角
- Prospective
入排标准
- 年龄范围
- 18 Years 至 —（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •Crohn's Disease at initial active stage with CDAI ≥
- •Starting Infliximab treatment
排除标准
- •Significant co-morbidity (e.g. cancer, intestinal tuberculosis, ischemic enteritis, radiation enteritis)
- •Other immunological disease (e.g. diabetes, rheumatoid arthritis systemic lupus erythematosus)
- •Current treatment with glucocorticoids, immunosuppressants and biological agents
- •Healthy controls
- •Inclusion Criteria:
- •No current disease
- •No daily drug use
- •Exclusion Criteria:
- •Significant co-morbidity (e.g. cancer, intestinal tuberculosis, ischemic enteritis, radiation enteritis)
- •Other immunological disease (e.g. diabetes, rheumatoid arthritis systemic lupus erythematosus)
- •Current treatment with glucocorticoids, immunosuppressants and biological agents
研究组 & 干预措施
CD patients
CD patients about to start Infliximab treatment, gives as i.v. injection of 5 mg/kg at baseline, after 2 weeks, 6 weeks, and then every 8th week.CD patients are followed for 14 weeks after the first administration of infliximab. At week 14 of Infliximab treatment, CD patients are classified as remission group (CDAI<150 and endoscopic mucosal healing, R group) and non-remission group (CDAI≥150 and/or mucosal non-healing group, N group).
干预措施: Infliximab (Drug)
结局指标
主要结局
Change of the frequency of Breg in N group
时间窗: week 0 of Infliximab treatment, week 14 of Infliximab treatment
The frequency of Breg is measured in peripheral blood by flow cytometry.
Change of the frequency of Breg in R group
时间窗: week 0 of Infliximab treatment, week 14 of Infliximab treatment
The frequency of Breg is measured in peripheral blood by flow cytometry.
Change of the frequency of Treg in R group
时间窗: week 0 of Infliximab treatment, week 14 of Infliximab treatment
The frequency of Treg is measured in peripheral blood by flow cytometry.
Change of the frequency of Treg in N group
时间窗: week 0 of Infliximab treatment, week 14 of Infliximab treatment
The frequency of Treg is measured in peripheral blood by flow cytometry.
次要结局
- Change of leucocyte count (WBC) in R group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of C-reactive protein (CRP) in R group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of platelet count (PLT) in R group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of platelet count (PLT) in N group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of erythrocyte sedimentation rate (ESR) in N group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of Crohn's disease activity index (CDAI) in R group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of erythrocyte sedimentation rate (ESR) in R group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of Crohn's disease activity index (CDAI) in N group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of C-reactive protein (CRP) in N group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)
- Change of leucocyte count (WBC) in N group(week 0 of Infliximab treatment, week 14 of Infliximab treatment)