Determining the Difference Between Circulating Follicular Regulatory T-cells and Follicular Helper T-cells in Subjects with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome-cells by Flow Cytometry
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Authors
Azim, Ambreen A.
Issue Date
2018
Type
Thesis
Language
en_US
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Abstract
The pathogenesis of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is currently unknown; however, B cell dysregulation has been suggested to be involved. It is well known that balance between follicular helper T cells (TFH) and follicular regulatory T cells (TFR) is important in the regulation of B cell responses. The purpose of this study is to develop methodology to accurately and reproducibly evaluate circulating TFR and TFH in ME/CFS cases and compare this to healthy controls. Immunophenotyping kits from Beckman Coulter, Thermo Fisher and BD Biosciences were tested using the protocol adapted from Wang et al. [1]. The final results show that the kit from BD Biosciences was the best performing kit. The kit from Beckman Coulter showed less antibody binding to the nuclear protein Foxp3, which is used to identify T-regulatory cells (Tregs). Also, the kit from Thermo Fisher did not show significant differences between stimulated cells and unstimulated cells. With the finalized protocol validated, future testing will begin using peripheral blood (PBMCs) from 20 ME/CFS cases and 20 healthy controls. These donors will be examined for the frequencies of circulating TFH and TFR by flow cytometry. Alterations in circulating TFH and TFR cells shift the balance from immune tolerance to immune responsive state, potentially contributing to deregulation of B cell immunity and the pathogenesis of ME/CFS. We hypothesize that there will be a decrease in the frequency of Treg cells, particularly CD45RA?FoxP3? Treg cells in ME/CFS patients when compared to healthy controls.
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In Copyright