Cryptococcal meningitis is one of the commonest causes of death in patients with AIDS and is associated with up to 500,000 deaths each year in Africa alone. A large proportion of patients die from the infection, in part because the current recommended treatment, amphotericin B for 2 weeks, is difficult to give in hospitals in the developing world, because it is relatively expensive and needs to be given intravenously and has side effects, often starting in the second week, meaning monitoring is needed with frequent blood tests. The alternative oral tablet treatment, fluconazole, that is available and cheap and currently commonly used, is much less effective. Therefore, based on a number of earlier small trials by the study team, we wish to test 2 new treatments, (1) Short, 1-week amphotericin B, and (2) Combination tablet treatment with high dose fluconazole plus another drug called flucytosine, that are as fast and effective in killing the infection as 2 weeks of amphotericin B. We will compare these new treatments with 2 weeks amphotericin B, in a larger, randomised trial that will enable us to see whether they are as good in preventing deaths from the infection. After 2 weeks of the study treatments, all patients will receive the usual follow on therapy with fluconazole, and will be started on drugs for the underlying HIV infection, as currently recommended, and followed up for 10 weeks. 570 patients (190 given each alternative treatment) will be studied. This is the minimum number needed to reliably compare the results of the treatments. The project has been developed with doctors in 3 centres in Malawi and Zambia where there are many cases and where alternative, affordable and practical treatments are urgently needed. Each centre has the laboratories needed, and experience in doing such trials. Both test treatments have been shown to be much more rapidly effective than fluconazole alone, and would have less side effects and be much more easily given in developing countries than 2 weeks amphotericin B. However, if 2 weeks amphotericin B was found to be the best treatment, then the costs required for its use could be justified. The costs as well as the effectiveness of the treatments will be compared to help decide which treatment to recommend in the future.

A thin continuous layer of cells covers the inside of all our blood vessels, separating blood from our tissues and cells. Unlike other cells in the body, blood does not clot when in contact with healthy endothelial cells. This unique and vital property stems from the secretion of molecules that prevent blood coagulation, and that rapidly dissolve blood clots if they do form. The endothelium is not however a one-trick-pony; in addition to controlling blood coagulation, they play essential roles in regulating blood flow, tissue repair and growth, and inflammation within the vasculature and adjacent tissues. Endothelial cells control all of these different processes through the secretion of a large number of different molecules. In effect, the vascular endothelium functions as a highly specialised multi-tasking secretory machine that responds rapidly to changes in its local environment. Alterations to the normal secretory function of endothelial cells are though to contribute to an increased risk of hypertension and atherosclerosis; major causes of stroke and heart attacks. Our research is aimed at trying to understanding the cellular mechanisms that regulate secretion from these cells. We focus particularly on the secretion of peptides and proteins involved in the control of blood coagulation (von Willebrand factor and tissue plasminogen activator) and inflammation (p-selectin and small cytokines such as interleukin-8). We combine biochemical, molecular, cell biological and biophysical approaches to directly analyse the synthesis, storage and secretion of these molecules in single endothelial cells. Understanding how the secretory function of endothelial cells is controlled under normal conditions will shed light on the changes that occur during disease and help us to develop new strategies for the treatment of vascular disorders

Lay Summary Understanding the genetic basis of disease involves ascribing gene mutations directly to a deleterious effect. Technological advances in whole genome sequencing now provide us with a plethora of information linking genetics to disease. An emerging bottleneck in translating that information to curing disease is a more complete understanding of how cells affected by disease interact in the body. Broadly, genetic disease can be caused at the level of the cell by gene mutation(s) preventing a certain cell-type from doing its job correctly. In the body, cells interact in 3 dimensions in a dynamic way within tissues and organs but also potentially send signals throughout the body. For example, neurons in the brain pass signals through complex local circuits that are initiated remotely and have consequences remotely. This circuitry can be damaged by neurological disease that can result in wide-ranging affects such as in Huntington's disease. However, the brain is not made up wholly of neurons and many neurological diseases are caused by dysfunction in other cells such as microglia, astrocytes and oligodendrocytes. This is exemplified by the now clear understanding that Multiple Sclerosis (MS) is caused by defects in oligodendrocyte function. Great strides have been made recently in the understanding of the cellular events that underlie MS resulting in novel drugs being trialed to treat the disease. We have developed new technologies and techniques to unravel the way that cells communicate with each other in living animals as disease develops. This study is focused on investigating type 2 Gaucher's disease (GD) that severely affects the brain but the tools and technologies involved would be broadly applicable to the study of virtually any type of cell in any tissue of the body. For the first time, we will combine the ability to generate neurons (but we can generate virtually any cell type) from mouse or human stem cells in the lab and genetically manipulate them so they emit quantifiable light when stimulated. Once these cells have been proven to respond to such stimuli we will engraft these normal reporter neurons into the brains of mice affected by GD before they are born. These experiments have been designed so as disease starts to affect the mice, the injected normal neurons will send signals by emitting light that will tell us how the cells are behaving and how they are interacting with cells affected by disease. Scientific Abstract We aim to combine for the first time induced pluripotent stem cell (iPSc) technologies with next generation light-emitting reporters to gain new insights into the cell:cell interactions that underlie disease progression in living animals. The use of luciferase luminescence for continued bioimaging of engrafted cells has already resulted in increased data output in substantially reduced cohorts of animals to track cell fate and distribution. This has so far been largely restricted to models of tumorigenesis and stem cell tracking. Here, we propose to use dual reporter cell lines generated from mouse iPSc to not only track cell fate but also transcription factor activity within these cells to provide new insights into how the engrafted cells interact with the local environment. For this focused study we will intracranially inject normal dual reporter neural stem cells (NSC) into fetal Gba1 knockout mice; a model of type 2 neuronopathic Gaucher's Disease and serially image for both reporters within the 14 day window after which most GD mice are sacrificed at a humane endpoint. We will also compare the reverse i.e. Gba1 knockout NSC into normal mice to determine if it is primarily the neural cells or resident activated microglia that are primarily responsible for neural degeneration. The technology being developed is not brain specific but is broadly applicable to many cell-types in many disease settings.