The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm Trichuris muris has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of T. muris. We identify 148 proteins secreted by T. muris and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. muris gene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted by T. muris provides important information on whipworm–host communication and forms the basis for future studies.

BackgroundThe protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite’s sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission.ResultsComparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered.ConclusionsThe need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1298-6) contains supplementary material, which is available to authorized users.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite–host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Parasitic helminths have coevolved with humans over millennia, intricately refining and developing an array of mechanisms to suppress or skew the host's immune system, thereby promoting their long-term survival. Some helminths, such as hookworms, cause little to no overt pathology when present in modest numbers and may even confer benefits to their human host. To exploit this evolutionary phenomenon, clinical trials of human helminth infection have been established and assessed for safety and efficacy for a range of immune dysfunction diseases and have yielded mixed outcomes. Studies of live helminth therapy in mice and larger animals have convincingly shown that helminths and their excretory/secretory products possess anti-inflammatory drug-like properties and represent an untapped pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins, glycans, post-translational modifications, and various metabolites. Although the concept of helminth-inspired therapies holds promise, it also presents a challenge to the drug development community, which is generally unfamiliar with foreign biologics that do not behave like antibodies. Identification and characterization of helminth molecules and vesicles and the molecular pathways they target in the host present a unique opportunity to develop tailored drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even so, much work remains to mine and assess this out-of-the-box therapeutic modality. Industry-based organizations need to consider long-haul investments aimed at unraveling and exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries with an eye on rapid and profitable turnarounds.

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Helminth parasites (worms) have evolved a vast array of strategies to manipulate their vertebrate hosts. Extracellular vesicles (EVs) are secreted by all helminth species investigated thus far, and their salient roles in parasite-host interactions are being revealed. Parasite EVs directly interact with various cell types from their hosts, including immune cells, and roles for their molecular cargo in both regulation and promotion of inflammation in the host have been reported. Despite the growing body of literature on helminth EVs, limited availability of genetic manipulation tools for helminth research has precluded detailed investigation of specific molecular interactions between parasite EVs and host target cells. Here, we review the current state of the field and discuss innovative strategies targeting helminth EVs for the discovery and development of new therapeutic strategies, placing particular emphasis on both anti-helminth vaccines and EV small RNAs for treating noninfectious inflammatory diseases.

BackgroundCanine babesiosis, caused by Babesia canis, is a prevalent and clinically relevant disease in Europe. Severe acute babesiosis is characterized by a high mortality but prognosis is not always correlated with clinical signs nor with the level of parasitemia.ObjectiveThis study evaluated prognostic markers associated with poor outcomes in acute Babesia canis infections.Animals and MethodsWe compared the results of routine laboratory profiles, hand‐held lactate and glucose analyzer, and the acute phase response in 2 groups of naturally infected dogs (7 survivors and 8 nonsurvivors). Samples were collected at the time of first admission and before any treatment. Subsequently, the course of prognostic markers was followed in 3 dogs experimentally inoculated with B. canis.ResultsNonsurvivors showed significantly higher concentrations of lactate, triglycerides and phosphate and lower hematocrit, leukocyte counts, total serum protein concentrations, and thrombocyte counts when compared to survivors. All nonsurvivors (8/8) had hyperlactatemia, whereas most survivors (6/7) had values within the reference range. All survivors had leucocyte counts within the reference range, unlike the nonsurvivors, which showed leukopenia. During the course of acute babesiosis, the variables serum lactate, triglyceride, and phosphate concentrations, and thrombocyte count only exceeded a prognostic threshold during acute crisis.Conclusions and clinical importancePoor outcome in acute B. canis infection is indicated by changes in the laboratory profile. Intensive care should be considered for dogs presenting with moderate anemia, severe thrombocytopenia, mild to moderate leukopenia, hyperlactatemia, moderately increased serum phosphate, and triglyceride concentrations, and moderately decreased total serum protein concentrations.