Association of Breg and Treg With the Clinical Effects of Infliximab in the Treatment of Patients With Crohn's Disease

Crohn Disease Clinical Trial

Official title:

Association of Breg and Treg With the Clinical Effects of Infliximab in the Treatment of Patients With Crohn's Disease

Clinical Trial Details — Status: Completed

Administrative data

• NCT number: NCT04272788
• Other study ID #: SAHoWMU-CR2020-01-205
• Status: Completed
• Start date: January 24, 2017
• Est. completion date: September 29, 2019

Study information

• Verified date: February 2020
• Source: Second Affiliated Hospital of Wenzhou Medical University
• Contact: n/a
• Is FDA regulated: No
• Study type: Observational

Clinical Trial Summary

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.

Description:

1. 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.

2. 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.

3. 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).

4. 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.

5. 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.

6. 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.

7. 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.

Recruitment information / eligibility

• Status: Completed
• Enrollment: 32
• Est. completion date: September 29, 2019
• Est. primary completion date: September 29, 2019
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years and older

Eligibility

CD patients

Inclusion Criteria:

1. Crohn's Disease at initial active stage with CDAI =150.

2. Starting Infliximab treatment

Exclusion Criteria:

1. Significant co-morbidity (e.g. cancer, intestinal tuberculosis, ischemic enteritis, radiation enteritis)

2. Other immunological disease (e.g. diabetes, rheumatoid arthritis systemic lupus erythematosus)

3. Current treatment with glucocorticoids, immunosuppressants and biological agents

Healthy controls

Inclusion Criteria:

1. No current disease

2. No daily drug use

Exclusion Criteria:

1. Significant co-morbidity (e.g. cancer, intestinal tuberculosis, ischemic enteritis, radiation enteritis)

2. Other immunological disease (e.g. diabetes, rheumatoid arthritis systemic lupus erythematosus)

3. Current treatment with glucocorticoids, immunosuppressants and biological agents

Related Conditions & MeSH terms

• Crohn Disease

Intervention

Drug:
• Infliximab
The CD patients at initial active stage without treatment of glucocorticoids, immunosuppressants and biological agents are included in the study when the decision to treat with Infliximab is already made. This study is observational, and all treatment and clinical follow-up are according to national guidelines.

Locations

Country	Name	City	State
China	SAHWenzhouMU	Wenzhou	Zhejiang

Sponsors (1)

Lead Sponsor	Collaborator
Second Affiliated Hospital of Wenzhou Medical University

Country where clinical trial is conducted

China, 

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Change of the frequency of Treg in R group	The frequency of Treg is measured in peripheral blood by flow cytometry.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Primary	Change of the frequency of Breg in R group	The frequency of Breg is measured in peripheral blood by flow cytometry.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Primary	Change of the frequency of Treg in N group	The frequency of Treg is measured in peripheral blood by flow cytometry.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Primary	Change of the frequency of Breg in N group	The frequency of Breg is measured in peripheral blood by flow cytometry.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of Crohn's disease activity index (CDAI) in R group	CDAI is a measure of Crohn's Disease severity. CDAI < 150 is in remission stage, and = 150 is in active stage. CDAI is negatively correlated with disease activity in CD patients.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of C-reactive protein (CRP) in R group	CRP is often combined to evaluate the disease activity of CD patients.The normal CRP range is (0-8) mg/L.Increased CRP indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of leucocyte count (WBC) in R group	WBC is often combined to evaluate the disease activity of CD patients.The normal WBC range is (4-10)×109/L.Increased WBC indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of platelet count (PLT) in R group	PLT is often combined to evaluate the disease activity of CD patients.The normal PLT range is (100-300)×109/L.Increased PLT indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of erythrocyte sedimentation rate (ESR) in R group	ESR is often combined to evaluate the disease activity of CD patients.The normal ESR range is (0-15)mm/h.Increased ESR indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of Crohn's disease activity index (CDAI) in N group	CDAI is a measure of Crohn's Disease severity. CDAI < 150 is in remission stage, and = 150 is in active stage. CDAI is negatively correlated with disease activity in CD patients.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of C-reactive protein (CRP) in N group	CRP is often combined to evaluate the disease activity of CD patients.The normal CRP range is (0-8) mg/L.Increased CRP indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of leucocyte count (WBC) in N group	WBC is often combined to evaluate the disease activity of CD patients.The normal WBC range is (4-10)×109/L.Increased WBC indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of platelet count (PLT) in N group	PLT is often combined to evaluate the disease activity of CD patients.The normal PLT range is (100-300)×109/L.Increased PLT indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment
Secondary	Change of erythrocyte sedimentation rate (ESR) in N group	ESR is often combined to evaluate the disease activity of CD patients.The normal ESR range is (0-15)mm/h.Increased ESR indicates disease activity.	week 0 of Infliximab treatment, week 14 of Infliximab treatment