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Effects of Thymoglobulin on Human B Cells

Yalnız qeydiyyatdan keçmiş istifadəçilər məqalələri tərcümə edə bilərlər
Giriş / Qeydiyyatdan keçin
Bağlantı panoya saxlanılır
Status
Sponsorlar
Northwestern University
Əməkdaşlar
Sanofi

Açar sözlər

Mücərrəd

This study is being done to better understand how immunosuppressive medications (anti-rejection medications that may or may not be approved by the Food and Drug Administration (FDA)) affect the alloimmune response (how a person's immune system reacts against another person's immune system). This will be determined by the blood tests run on the patients donated blood cells.
Specifically, the investigators will test different immunosuppression medications using in-vitro assays (in the laboratory, in test tubes) alone and/or in combination to test how they can affect B-cell proliferation and differentiation, leading to an altered distribution among defined B-cell subsets and to study if exposure of B cells to thymoglobulin can have effects on subsequent interaction between B and T cell in vitro.
To enable the investigators to understand how immunosuppressant medications affect immune systems in transplant patients, the investigators need to understand how they affect immune systems in healthy people. To do this, the investigators will need to study blood collected from healthy volunteers.

Təsvir

This is a research study of healthy subjects who will furnish blood samples for various assays over a period of one year.

Peripheral blood mononuclear cells (PBMC) will be isolated from healthy volunteers and CD19+ B cells will be selected using CD19 magnetic micro-beads.

B-cell cultures will be performed in culture medium consisting of Iscove's MDM (IMDM) medium (Gibco, Paisley, UK) supplemented with 10% human serum (HNS- GemCell), 0.5 ml Human Insulin (Sigma), 200mM L-Glutamine (Sigma) and 0.5% Gentamycin (Gibco).

Total CD19+B cells will be cultured in the presence of anti-IgM (3μg/ml), anti-CD40 (100ng/ml), IL-21 (100ng/ml) (BCR/CD40-stimulation) for 6 days in culture plates 96 wells round bottom (1.0x105 cells/well), and Thymoglobulin at different concentrations (10 to 100 μg/ml) with or without calcineurin inhibitor (Tacrolimus, TAC) and mTOR inhibitor (Sirolimus, SRL) will be added at day 0.

A variety of parameters of B cell activity including proliferation, activation, differentiation and cytokine production will be monitored by flow cytometry.These experiments are further explained in the outcome measures section below.

The investigators will conduct univariate and bivariate analyses for all variables. The investigators will evaluate continuous variables using t-tests or F-tests and categorical variables using Chi-square or Fisher Exact tests. The investigators will examine data distributions and test all variables for linear relationships or non-linear relationships. Descriptive statistics (means, standard deviation, frequency) and correlations (both Pearson and Spearman) among variables will be conducted to assess the data. Necessary transformation and imputations will be conducted based on the raw data distribution. All data analyses will be performed using SAS 9.3 statistical software (SAS Inc., Cary, NC). All tests will be two-sided and an error rate of α <0.05 will be considered statistically significant.

Tarixlər

Son Doğrulandı: 02/28/2018
İlk təqdim: 04/29/2014
Təxmini qeydiyyat təqdim edildi: 05/11/2014
İlk Göndərmə: 05/13/2014
Son Yeniləmə Göndərildi: 03/26/2018
Son Yeniləmə Göndərildi: 03/28/2018
Həqiqi Təhsilin Başlama Tarixi: 03/31/2014
Təxmini İlkin Tamamlanma Tarixi: 02/28/2018
Təxmini İşin Tamamlanma Tarixi: 02/28/2018

Vəziyyət və ya xəstəlik

Acute Humoral Rejections
Chronic Humoral Rejections
Transplant

Müdaxilə / müalicə

Procedure: Blood Draws

Faza

-

Uyğunluq Kriteriyaları

Təhsil üçün uyğun yaşlar 18 Years Üçün 18 Years
Təhsilə Uyğun CinslərAll
Nümunə götürmə metoduNon-Probability Sample
Sağlam Könüllüləri qəbul edirBəli
Kriteriyalar

Inclusion Criteria:

The patient:

1. should be more than 18 years old,

2. should weigh more than 110 lbs and

3. should not be pregnant at the time of blood donation.

Exclusion Criteria:

(1) Not fitting within inclusion criteria

Nəticə

İlkin nəticə tədbirləri

1. CD19+B cell division [12 days]

3-6x106 cells of interest will be labeled with 1.5μM CFSE before stimulation. After 6 days, B cells will be recovered, washed, counted and used to define marker settings. The cell phenotype will be analyzed by five-color flow cytometry (FC500, Beckman-Coulter) and by 13-color flow cytometry (Fortessa) and data will be analyzed using CXP software. The supernatant of these B-cell cultures will be using for the cytokines detection, and to determine the number of IgM and IgG producing cells by Luminex.At day 6, cells will be re-stimulated with phorbol 12-myristate 13-acetate (PMA) and Ionomycin, and analyzed by intracellular cytokine staining. Total CD19+ B cells will be sorted into a CD27- (naïve) and CD27+ (memory) B-cell fraction and the phenotypic characterization (assessed by flow cytometry) of these cells will be showed after 6 days of culture with anti-IgM (5μg/ml), anti-CD40 (100ng/ml), and IL-21 (100ng/ml) in the presence or absence of the drugs.

İkincili Nəticə Tədbirləri

1. proliferated T-cells [6 days]

The investigators will co-culture B cells (pre-stimulated with α-IgM and α-CD40 mAb plus IL-21 for 6 days in the presence and absence of Thymoglobulin) with allo-CD4+CD25- T cells (ratio 2:1). After 6 days the investigators will analyze the % of proliferated T-cells and the cytokine production.

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