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Abstract Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells

Invariant Natural Killer T (iNKT) cells are key players in the immunity to several pathogens; however, their involvement in the resistance to Paracoccidioides brasiliensis infection remains unknown. Using splenocytes from CD1d (CD1d-/-) and iNKT-deficient (Jα18-/-) mice, we found that iNKT cells are the innate source of IFN-γ after P. brasiliensis infection and are required to potentiate macrophage oxidative burst and control fungal growth. To determine whether iNKT cells contribute in vivo to host resistance against P. brasiliensis infection, we infected intratracheally wild-type and Jα18-/- C57BL/6 mouse strains with the virulent Pb18 isolate. iNKT cell deficiency impaired the airway acute inflammatory response, resulting in decreased airway neutrophilia and reduced IFN-γ, KC, and nitric oxide (NO) production. The deficient innate immune response of Jα18-/- mice to Pb18 infection resulted in increased fungal burden in the lungs and spleen. Besides, the activation of iNKT cells in vivo by administration of the exogenous iNKT ligand α-galactosylceramide (α-GalCer) improved host resistance to P. brasiliensis infection. Although the mechanisms responsible for this phenomenon remain to be clarified, α-GalCer treatment boosted the local inflammatory response and reduced pulmonary fungal burden. In conclusion, our study is the first evidence that iNKT cells are important for the protective immunity to P. brasiliensis infection and their activation by an exogenous ligand is sufficient to improve the host resistance to this fungal infection.

All extracellular forms of Trypanosoma cruzi, the causative agent of Chagas disease, release extracellular vesicles (EVs) containing major surface molecules of the parasite. EV release depends on several mechanisms (internal and external). However, most of the environmental conditions affecting this phenomenon are still unknown. In this work, we evaluated EV release under different stress conditions and their ability to be internalized by the parasites. In addition, we investigated whether the release conditions would affect their immunomodulatory properties in preactivated bone marrow-derived macrophages (BMDM). Sodium azide and methyl-cyclo-β-dextrin (CDB) reduced EV release, indicating that this phenomenon relies on membrane organization. EV release was increased at low temperatures (4°C) and acidic conditions (pH 5.0). Under this pH, trypomastigotes differentiated into amastigotes. EVs are rapidly liberated and reabsorbed by the trypomastigotes in a concentration-dependent manner. Nitrosative stress caused by sodium nitrite in acid medium or S-nitrosoglutathione also stimulated the secretion of EVs. EVs released under all stress conditions also maintained their proinflammatory activity and increased the expression of iNOS, Arg 1, IL-12, and IL-23 genes in IFN-γ and LPS preactivated BMDM. In conclusion, our results suggest a budding mechanism of release, dependent on the membrane structure and parasite integrity. Stress conditions did not affect functional properties of EVs during interaction with host cells. EV release variations under stress conditions may be a physiological response against environmental changes.

In the past years, extracellular vesicles (EVs) have become an important field of research since EVs have been found to play a central role in biological processes. In pathogens, EVs are involved in several events during the host–pathogen interaction, including invasion, immunomodulation, and pathology as well as parasite–parasite communication. In this report, we summarised the role of EVs in infections caused by viruses, bacteria, fungi, protozoa, and helminths based on the talks and discussions carried out during the International Society for Extracellular Vesicles (ISEV) workshop held in São Paulo (November, 2016), Brazil, entitled Cross-organism Communication by Extracellular Vesicles: Hosts, Microbes and Parasites.

Depending on Leishmania species and the presence/absence of virulence factors, Leishmania extracellular vesicles (EVs) can differently stimulate host immune cells. This work is aimed at characterizing and evaluating the protective role of EVs released by Leishmania amazonensis promastigotes under different maintenance conditions. Initially, using a control strain, we standardized 26°C as the best release temperature to obtain EVs with a potential protective role in the experimental leishmaniasis model. Then, long-term (LT-P) promastigotes of L. amazonensis were obtained after long-term in vitro culture (100 in vitro passages). In vivo-derived (IVD-P) promastigotes of L. amazonensis were selected after 3 consecutive experimental infections in BALB/c mice. Those strains developed similar lesion sizes except for IVD-P at 8 weeks post infection. No differences in EV production were detected in both strains. However, the presence of LPG between LT-P and IVD-P EVs was different. Groups of mice immunized with EVs emulsified in the adjuvant and challenged with IVD-P parasites showed decreased lesion size and parasitic load compared with the nonimmunized groups. The immunization regimen with two doses showed high IFN-γ and IgG2a titers in challenged mice with either IVD-P or LT-P EVs. IL-4 and IL-10 were detected in immunized mice, suggesting a mixed Th1/Th2 profile. EVs released by either IVD-P or LT-P induced a partial protective effect in an immunization model. Thus, our results uncover a potential protective role of EVs from L. amazonensis for cutaneous leishmaniasis. Moreover, long-term maintenance under in vitro conditions did not seem to affect EV release and their immunization properties in mice.

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.