High accuracy 4D cell tracking into explanted skin using two-photon excitation microscopy.

Two-photon excitation microscopy (2PEM) analysis of large explanted organs is still laborious, principally because of tissue movements inducing lateral and axial drifts during extended imaging sessions. Here, we describe a two-step approach to track motile T cells in murine dorsal explanted skin with the best accuracy. First, we compared various explanted skin mounting methods for 2PEM analysis to define the setup allowing for minimal sample drift over time. Second, we developed two algorithms with the ImageJ software (National Institute of Health, Bethesda, MD) to correct the residual drift using lateral and axial registration of the collagen network. Finally, we applied the macro we developed to track fluorescent T cells in explanted skin. We found that our newly developed macro is more efficient than freely or commercially available software for shift correction, leading to more accurate velocity calculations. Our work provides a practical guide for investigators interested to employ skin-imaging approaches and offers a free alternative to commercial software for correcting lateral and axial drifts.

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow.

BACKGROUND: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. OBJECTIVES: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. RESULTS: Videomicroscopy of DIOC-labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P-selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvß3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y-27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.

Detection of human cytomegalovirus genome and gene products in central nervous system tumours.

Human cytomegalovirus (HCMV) genome and related proteins have been reported in a great proportion of malignant gliomas. However, these results are unexpected since HCMV is not known as an oncogenic virus. By immunohistochemistry (with an anti-IE1 monoclonal antibody) and in situ hybridisation (with biotinylated DNA probes) on tissue microarrays and frozen sections, we investigated a French series of central nervous system (CNS) tumours, including 97 glioblastomas. In 10 cases of glioblastoma, rare astrocyte-like cells, admixed with tumour cells, stained positively for HCMV and in one case a doubtful staining of rare cells was noticed. This may indicate a reactivation of the virus under local immunosuppression but none of the cases of CNS tumours (n=132) contained HCMV genomes and/or proteins in a significant proportion of tumour cells. Our results strongly suggest that HCMV is unlikely to be implicated in the development of human malignant gliomas, at least in French cases.

Incoming human cytomegalovirus pp65 (UL83) contained in apoptotic infected fibroblasts is cross-presented to CD8(+) T cells by dendritic cells.

Human cytomegalovirus (HCMV) infection is well controlled mainly by cytotoxic CD8(+) T lymphocytes (CTL) directed against the matrix protein pp65 despite the numerous immune escape mechanisms developed by the virus. Dendritic cells (DCs) are key antigen-presenting cells for the generation of an immune response which have the capacity to acquire antigens via endocytosis of apoptotic cells and thus present peptides to major histocompatibility complex class I-restricted T cells. We examined whether this mechanism could contribute to the activation of anti-pp65 CTL. In this study, we show that infection by HCMV AD169 induced sensitization of MRC5 fibroblasts to tumor necrosis factor alpha-mediated apoptosis very early after virus inoculation and that pp65 contained in apoptotic cells came from the delivery of the matrix protein into the cell. We observed that immature DCs derived from peripheral monocytes were not permissive to HCMV AD169 infection but were able to internalize pp65-positive apoptotic infected MRC5 cells. We then demonstrated that following exposure to these apoptotic bodies, DCs could activate HLA-A2- or HLA-B35-restricted anti-pp65 CTL, suggesting that they acquired and processed properly fibroblast-derived pp65. Together, our data suggest that cross-presentation of incoming pp65 contained in apoptotic cells may provide a quick and efficient way to prime anti-HCMV CD8(+) T cells.