Sascha Keller

BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. METHOD: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. RESULTS: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. CONCLUSIONS: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics.

BACKGROUND: Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells. METHODS: SKOV3 exosomes were labelled with carboxyfluorescein diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts. RESULTS: In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-lactose reduced exosomes uptake to a comparable extent as the control D-glucose. CONCLUSIONS: In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.