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Title: Measurement of cell proliferation using in vivo stable isotope labelling : application to T-cell homeostasis in young and elderly healthy human subjects
Author: Ahmed, Raya
ISNI:       0000 0004 7427 0513
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2015
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Immune homeostasis is achieved through a long-term balance between proliferation and cell death. With ageing of the immune system, immunosenescence, dysfunctional highly differentiated (CD57high CD28|OW) T-cells accumulate. Their presence correlates with functional impairments which may contribute to the increased risk of infectious and age-related diseases in an expanding elderly population. Whether such cells proliferate in vivo is contentious. Answering that question is the focus of this thesis, but methodological aspects of measurement of cell proliferation by either heavy water (D2O) or deuterium-labelled (deuterated) glucose needed to be addressed first as, historically, they appear to give different results. This thesis describes a series of experiments: (i) To identify the source of the disparity between heavy water or deuterated- glucose, using in vitro tissue culture and in vivo murine models, and (ii) To investigate the kinetics of senescent T-cells and T-cell memory precursors (stem cell memory cells, TScm) in healthy young and elderly subjects. In vitro experiments were conducted using Jurkat cells labelled with deuterated-glucose and/or heavy water. In Murine in vivo experiments, mice received liquid feed labelled with either heavy water or deuterated-glucose. Disparities in lymphocyte proliferation rate estimates were the consequence of different normalisation approaches and were resolved when similar normalisation was applied. Eight healthy human subjects (4 young and 4 elderly, all CMV-seropositive) were given heavy water orally for seven weeks. T-cell subsets were sorted by flow cytometry and analysed for DNA deuterium content by gas-chromatography mass-spectrometry to derive modelled proliferation rates. We found that: (i) CD57+ "senescent" cells (CD4 and CD8) retain proliferative capacity, and have relatively short life-spans (high turnover). (ii) Tscm represent a rapidly-dividing subpopulation (iii) Similar patterns were observed in young and elderly These studies demonstrate how isotope-based measurements may be applied to decipher T- cell differentiation and homeostasis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available