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

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.

Study Overview

• Status: Completed
• Conditions: Crohn Disease
• Intervention / Treatment: Drug: Infliximab

Detailed 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.

• Study Type: Observational
• Enrollment (Actual): 32

Contacts and Locations

Study Locations

• China
  • Zhejiang
    • Wenzhou, Zhejiang, China
      • SAHWenzhouMU

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (ADULT, OLDER_ADULT)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

Patients from the gastroenterology department at the Second Affiliated Hospital of Wenzhou Medical University are eligible for entry.

Healthy controls are recruited at Health Examination Center of the Second Affiliated Hospital of Wenzhou Medical University.

Description

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

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
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).	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.
Healthy controls; Healthy controls without Crohn's Disease.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
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
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
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
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 Outcome Measures

Outcome Measure	Measure Description	Time Frame
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
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
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
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
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
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
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
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
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
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

Collaborators and Investigators

• Sponsor: Second Affiliated Hospital of Wenzhou Medical University
• Investigators: Principal Investigator: Yi Jiang, PhD, Second Affiliated Hospital of Wenzhou Medical University

Study record dates

Study Major Dates

• Study Start (ACTUAL): January 24, 2017
• Primary Completion (ACTUAL): September 29, 2019
• Study Completion (ACTUAL): September 29, 2019

Study Registration Dates

• First Submitted: February 11, 2020
• First Submitted That Met QC Criteria: February 13, 2020
• First Posted (ACTUAL): February 17, 2020

Study Record Updates

• Last Update Posted (ACTUAL): February 17, 2020
• Last Update Submitted That Met QC Criteria: February 13, 2020
• Last Verified: February 1, 2020

More Information

Terms related to this study

• Keywords: Crohn Disease; Infliximab; Treg; Breg
• Additional Relevant MeSH Terms: Digestive System Diseases; Gastrointestinal Diseases; Gastroenteritis; Intestinal Diseases; Inflammatory Bowel Diseases; Crohn Disease; Antirheumatic Agents; Gastrointestinal Agents; Dermatologic Agents; Infliximab
• Other Study ID Numbers: SAHoWMU-CR2020-01-205

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED
• IPD Plan Description: Data sets will be made available on relevant requests and in accordance with journal guidelines when publishing results from this study.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No