The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30–100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

The neutral amino acid carrier composition of luminal and abluminal membranes of the blood-brain barrier has been studied using isolated membrane vesicles. Phenylalanine was carried almost exclusively by a high affinity (Km = 10 +/- 2 microM), Na(+)-independent amino acid transport system, presumably L1 system, that was found to be symmetrically distributed between luminal and abluminal membranes. Inhibition of phenylalanine uptake was used to determine the affinities (Ki values) toward leucine (17 +/- 3 microM), tryptophan (8 +/- 1), 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH) (11 +/- 2), alanine (628 +/- 117), and glutamine (228 +/- 51). Alanine was found to be transported by two Na(+)-dependent transport systems that were located exclusively on the abluminal membrane. Kinetic and inhibition experiments indicated that one of these activities was due to system A, which is probably the main route for Na(+)-dependent alanine transport (Km = 0.6 +/- 0.2 mM) under physiological conditions. The other Na(+)-dependent activity was attributed to a B(o,+)-like system based on its sensitivity toward BCH. This latter system showed greater affinity for large neutral amino acids. The affinities of these two transport systems for several other amino acids were also studied.

Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that gamma-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5'-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Na+/K(+)-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K(+)-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.