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The properties and potential applications of human lymphoblastoid cell linen have been investigated. Buch lines have been established by others from Burkitt's lymphoma tissue, from leukaetnic patients, from those with infectious mononucleosis and latterly from healthy subjects. There is an association between EB virus (Herpes-like virus of Epstein and Barr) and the growth of human lymphob1astold cell lines in vitro. The same virus is linked sero-epidemiologically with Burkitt's lymphoma, nasopharyngeal carcinoma, infectious mononucleosis and a number of other diseases of man. The exact role of the virus both in vivo and in vitro is a matter of some controversy. Using a simple culture technique, 40 cell lines were established from the peripheral blood leukocytes of a diaqnostically rai3:»d group of patients, from normal cord blood and from one h althy adult. 25 of these cultures were initiated by adding EB virus (in the form of lethally irradiated cells of a virus-positive line) to the fresh blood leukocytes. Evidence from experiments of this type suggest that EB virus has a lyraphoproliferative action in vitro. The cell lines are morphologically etlmost indistinguishable. They resemble primitive cells of the lymphoid series and appear to have the property of indefinite growth in vitro. They include some phagocytic cells and immunoglobulins are released into the culture medium. Feu of the lines were chromosomally abnormal when examined shortly after establishment but all of those examined after more than 1 year of continuous growth had abnormal karyotypes. There was no evidence for the recurrence of any specific chromosome aberration in a significant proportion of the lines. Antigenleally the established lines differ from the original donor's fresh lymphocytes. This is shown by measurement of tritiated thymidine uptake by lymphocytes exposed in vitro to irradiated cells from an autochthonous line. Together with the activation process, the fresh lymphocytes acquire cytotoxic potential directed specifically against the cell line used in the activation phase. Tt is suggested that this system constitutes a model for an immunological surveillance mechanism operating in vivo to eliminate deviant cells. Cell lines of this type offer rcone for detailed investiga¬ tion of the induction of proliferation in human tissue (in this case probably by FB virus) and of the immunological processes by which altered autochthonous cells may be recognised and destroyed. Both of these topics are relevant to the basic study of malignant disease. The prolonged life span and relative stability of these lines in vitro also presents opportunities in the Field of human genetics, both for the investigation of inherited disorders of man and, experimentally, for the measurement of spontaneous and induced mutation rates, analysis of gene linkage and chromosome mapping.

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A significant release of catecholamines within the heart has been observed during myocardial ischaemia. Ischaemia-induced catecholamine release can be markedly inhibited by desipramine and other amine uptake blocking agents, allowing investigation of the importance of such release for arrhythmia production. The mechanism of this release appears to occur by a carrier-mediated efflux from neurones, which is not operative under normal conditions. The aim of the project has been to study this release process in chromaffin cells isolated from the bovine adrenal medulla, which are recognised as a model system for studying the sympathetic nervous system. Understanding this process of catecholamine release may lead to new methods of protecting the heart against ischaemia-induced arrhythmias. Isolated chromaffin cells could be maintained in primary culture for up to 7 days and retained all their normal secretory responses. Conditions designed to mimic ischaemia, that is, anoxia or metabolic inhibition, resulted in a significant release of catecholamines. This release was shown to be independent of extracellular calcium but, in contrast to the release observed in ischaemic hearts, it was not inhibited by uptake<SUB>1</SUB> blockers. One of the main criteria for exocytosis is the co-release of other secretory granule components. Polyacrylamide gel electrophoresis and Western blotting techniques were utilised to examine this following metabolic inhbition. Significant levels of the granule proteins chromogranin A, neuropeptide Y and ATP were measured following metabolic inhibition, indicative of an exocytotic mechanism. Furthermore, there was no release of the cytosolic protein lactate dehydrogenase, indicating that there was no breakdown of the cell membrane during metabolic inhibition. Over the first 10 minutes of metabolic inhibition there was a marked uptake of <SUP>22</SUP>Na<SUP>+</SUP> by the cells. It is suggested that this Na<SUP>+</SUP> influx triggers the catecholamine release by affecting the cytosolic Ca<SUP>2+</SUP> concentration through a direct effect on intracellular stores. Intracellular Ca<SUP>2+</SUP> mobilisation was measured using the calcium-sensitive fluorescent probe Fura-2. It was found that cytosolic free calcium levels were increased in response to metabolic inhibition. The conditions requird to evoke carrier-mediated efflux were also examined. Cytosolic levels of catecholarmines could be artificially raised following treatment with reserpine. Cytoplasmic catecholamine levels were measured following permeabilisation with the detergent digitonin which renders the plasma membrane leaky. Conditions designed to reverse the uptake carrier and cause carier-mediated efflux in the presence of raised cytoplasmic catecholamines such as removal of extracellular sodium, did not evoke any catecholamine overflow. These studies suggest the chromaffin cell uptake<SUB>1</SUB> carrier is not reversible and may be gated in some way. This work has, therefore, raised questions concerning the suitability of chromaffin cells as a conventional model for sympathetic nerve terminals.

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