The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.
KEYWORDS: Diffusion, Tissues, Two photon excitation microscopy, Pathogens, Microscopes, In vivo imaging, Signal detection, Physics, Microscopy, Inspection
Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells
(NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen
associated molecular pattern (PAMP) activated-DCs in inducing NK
cell-mediated anti-tumor responses.
Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected
with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4
dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory
conditions are also evaluated.
Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar
kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK
dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and
average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might
efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK
cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.
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