Use of venesected blood from patients with haemochromatosis to isolate and culture lmmune cells
Hypothesis: Interactions between DCs and sinusoidal endothelium are critical for the recruitment and differentiation of DC precursors and involve specific adhesion moleculesBackground: Dendritic cells respond rapidly to pathogens by taking up antigen and inducing primary immune responses. Newly generated DC precursors migrate from hone-marrow to .non-lymphoid organs where signals from invading pathogens stimulate them to take up and process antigen. Antigen-loaded DCs exit via lymphatics into draining lymph node where they stimulate T-cells. In the liver migration into lymph nodes occurs via a unique trans-sinusoidal pathway and interactions with sinusoidal endothelium will thus be critical both for the recruitment of circulating DC precursors into the liver and for their subsequent emigration into lymph node.Objectives: To determine the molecular regulation of DC interactions with hepatic sinusoidal endotheliumDesign: We will use in vitro cell culture models and flow-based adhesion assays to determine: 1) Whether DC precursors and mature DC show differential binding to sinusoidal endothelium and lymph node endothelium. 2) The molecular regulation of DC precursor recruitment via sinusoidal endothelium under flow. Flow-based adhesion assays allow us to study capture static adhesion and transmigration of DCs through sinusoidal endothelium under physiological conditions. The molecules involved will be determined using blocking antibodies. In particular we will concentrate on four novel molecules L-SIGN LYVE-l Endo-l80 and VAP-l that we have shown to be expressed on sinusoidal endothelium3) the molecules involved in reverse transmigration into the sinusoids. After being activated in tissue DCs migrate back into the sinusoids by a poorly understood process of reverse transmigration. We will model this in vitro and determine the adhesion molecules and chemokines involved. 4) The effect of transmigration or. DC function. Reverse transmigrated DCs will be collected and tested for their ability to stimulate 7 cell activation or apoptosis in response to antigen and allogeneic cells. We will determine whether the stimulated 7 cells die or proliferate and if the latter use intracellular cytokine and in vitro suppression assays to determine whether they are 7111 112 or regulatory cells. Outcomes: Little is known about the regulation of DC migration via endothelium although this will be critical for normal immune function as well as the pathogenesis of inflammatory liver disease. These studies will allow me to use a variety of in vitro approaches to define this process. The results will be of importance for understanding DC function and may s. . . Cont’d on Additional Pages.
|PI Name||Adams - DH|
|Sponsor||University Hospital Birmingham NHS Trust|
|Proposed End Date||31/12/2019|
|Study Run through CRF?||No|
|Recruitment so far||0|