Nucleic acid changes during lymphocyte transformation in vitro
Circulating blood lymphocytes (a) do not usually divide in vitro (Trowell,1965), and (b), are of importance in immunity (Gowans + McGregor,1965). Nowell in 1960 discovered that when a bean extract, PHA, was added to these cells in culture they were suddenly activated to transform and divide. Similar cytological changes are seen within lymphoid organs in the course of immune responses (Fagraeus, 1948; Scothorne,1957; Gowans,1962; Oort + Turk,1965). I have studied the nucleic acid changes occurring during in vitro transformation in order to gain some understanding of the cellular control mechanisms involved and of the immune significance of the phenomenon. The results of this study are presented from chapter IV onwards after initial chapters on the literature of lymphocyte transformation in vitro (chapter II), and on the methods which have been employed (chapter III). In chapter IV the quantitative nucleic acid changes occuring during transformation are described. Such measurements were designed to produce results which could be simply interpreted. The main questions which are asked are:- (a) How long will cells grow in culture without medium replenishment? (b) How much PHA must be added in order to obtain a given transformation response? (c) Is this amount of PHA proportional to the number of cells present, or the amount of serum in the culture? (d) How long must cultures be exposed to PHA in order that a subsequent transformation response will occur? However quantitative nucleic acid assay was not sufficiently sensitive to answer the further questions which arose, In chapter V measurements of the incorporation of [5-$^{3}$H] uridine during transformation are described. Such measurements produced results which were difficult to interpret. Most of the chapter is devoted to finding to what extent the incorporation of [5-$^{3}$H] uridine can be treated in simple terms. A description is given of a new phenomenon by which uridine itself, at certain critical concentrations, stimulates the incorporation of [5-$^{3}$H] uridine. Evidence that the initial stimulation of uridine incorporation by PHA is a true effect, and not an artifact caused by a change in pool size, is presented. In chapter VI the incorporation of [5-$^{3}$H] uridine is used to further examine the mechanism of initial activation of cells by PHA, and the relationship of attachment of cells to glass, to this activation. In chapter VII qualitative aspects of RNA synthesis are examined. The sucrose density gradient profiles of RNA labelled with [5-$^{3}$H] uridine at different times of culture are compared, The "messenger" activity of lymphocyte RNA in a sub-cellular system is examined, In chapter VIII the incorporation of [5-$^{3}$H] uridine is used to investigate the role of serum in the "mixed-cell" culture phenomenon, using cells and serum from homologous donors . Blood lymphocytes constantly recirculate from the blood to lymphoid tissues and so cannot be considered in isolation. In the appendix review sections on lymphoid tissues (appendix I), and their function (appendix II), are presented in an attempt to provide both a general framework against which the PHA work can be viewed, and an introduction to the final section of the appendix. This presents a theory of immunity which arose out of the PHA studies by a process of analogy, - PHA being considered (but not proven), to be a "non-specific" antigen. In so far as the thesis can be said to have any underlying theme it is the following. The response of a specific cell to a specific antigen should be detectable either by examining the metabolic changes occurring in the cell to prepare it for protein secretion and cell division, or by measuring the actual antibody synthesised (see appendix ). Measurement of the initial RNA changes in an activated cell should be a way of detecting such activation shortly after it occurs. A study of early RNA changes in PHA-stimulated cells should throw some light on the problem of cell stimulation by an antigen, by providing, at the very least, some knowledge of the methodology entailed.
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