Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined.From the Clinical EditorThe authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.

Nucleotide-binding oligomerization domain-containing-2 (NOD2) acts as a bacterial sensor in dendritic cells (DCs), but it is not clear how bacterial recognition links with antigen presentation after NOD2 stimulation. NOD2 variants are associated with Crohn's disease, where breakdown in self-recognition of commensal bacteria leads to gastrointestinal inflammation. Here we show NOD2 triggering by muramyldipeptide induces autophagy in DCs. This effect requires receptor-interacting serine-threonine kinase-2 (RIPK-2), autophagy-related protein-5 (ATG5), ATG7 and ATG16L1 but not NLR family, pyrin domain containing-3 (NALP3).We show that NOD2-mediated autophagy is required for both bacterial handling and generation of major histocompatibility complex (MHC) class II antigen-specific CD4(+) T cell responses in DCs. DCs from individuals with Crohn's disease expressing Crohn's disease-associated NOD2 or ATG16L1 risk variants are defective in autophagy induction, bacterial trafficking and antigen presentation. Our findings link two Crohn's disease-associated susceptibility genes in a single functional pathway and reveal defects in this pathway in Crohn's disease DCs that could lead to bacterial persistence via impaired lysosomal destruction and immune mediated clearance.

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999) J. Cell Biol. 144, 789 -801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.The extracellular matrix glycosaminoglycan hyaluronan (HA) 1 is a large polymer of N-acetyl-D-glucosamine and D-glucuronic acid (molecular mass 10 5

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.