Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encase several types of molecules. Their contents mostly reflect their cell origin and possible targets at other locations in the organism and can be modified in pathological conditions to interfere with intercellular communication, thus promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make such vesicles ideal for biomarker discovery in human diseases. Here, we describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of chronic Chagas disease (CCD) patients. We illustrated this procedure by studying a population of patients with Chagas disease at the indeterminate chronic stage, in which the Trypanosoma cruzi is very scarce in circulation. EVs were harvested from blood collected without or with different anticoagulants. Protein and nanoparticle tracking analysis was used to measure EVs size and concentration. The EVs were purified by ultracentrifugation, followed by size-exclusion chromatography and characterized by chemiluminescent enzyme-linked immunosorbent assay and dot blot using antibodies that recognized parasite-derived EVs, such as hyperimmune sera, polyclonal and monoclonal antibodies against trans-sialidase and mucins. In parallel, antibodies against classical human EV markers CD9, CD63, CD81, and CD82, were also analyzed. The results showed that anticoagulants did not interfere with the analyzed parameters and circulating EVs from CCD patients contain T. cruzi antigens and classical human exosomal markers. Overall, our protocol is adequate for the isolation of the total circulating EVs and can serve as an important basis for further studies on biomarker discovery in Chagas' disease.

Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans.

The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host.

TLR9/MyD88/TRIF signaling activates host immune inhibitory CD200 in Leishmania infection.

Virulent protozoans named Leishmania in tropical and subtropical areas produce devastating diseases by exploiting host immune responses. Amastigotes of Leishmania amazonensis stimulate macrophages to express CD200, an immunomodulatory ligand, which binds to its cognate receptor (CD200R) and inhibits the inducible nitric oxide synthase and nitric oxide (iNOS/NO) signaling pathways, thereby promoting intracellular survival. However, the mechanisms underlying CD200 induction in macrophages remain largely unknown. Here, we show that phagocytosis-mediated internalization of L. amazonensis amastigotes following activation of endosomal TLR9/MyD88/TRIF signaling is critical for inducing CD200 in infected macrophages. We also demonstrate that Leishmania microvesicles containing DNA fragments activate TLR9-dependent CD200 expression, which inhibits the iNOS/NO pathway and modulates the course of L. amazonensis infection in vivo. These findings demonstrate that Leishmania exploits TLR-signaling pathways not only to inhibit macrophage microbicidal function, but also to evade host systemic immune responses, which has many implications in the severity of the disease.

Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and biological activity against Trypanosoma cruzi.

Chagas disease is a neglected protozoan disease that affects more than eight million people in developing countries. Due to the limited number and toxicity profiles of therapies in current use, new drugs are urgently needed. In previous studies, we reported the isolation of two related antitrypanosomal neolignans from Nectandra leucantha (Lauraceae). In this work, a semi-synthetic library of twenty-three neolignan derivatives was prepared to explore synthetically accessible structure activity relationships (SAR) against Trypanosoma cruzi. Five compounds demonstrated activity against trypomastigotes (IC50 values from 8 to 64 µM) and eight showed activity against intracellular amastigotes (IC50 values from 7 to 16 µM). Eighteen derivatives demonstrated no mammalian cytotoxicity up to 200 µM. The phenolic acetate derivative of natural dehydrodieugenol B was effective against both parasite forms and eliminated 100% of amastigotes inside macrophages. This compound caused rapid and intense depolarization of the mitochondrial membrane potential, with decreased levels of intracellular reactive oxygen species being observed. Fluorescence assays demonstrated that this derivative affected neither the permeability nor the electric potential of the parasitic plasma membrane, an effect also corroborated by scanning electron microscopy studies. Structure-activity relationship studies (SARs) demonstrated that the presence of at least one allyl side chain on the biaryl ether core was important for antitrypanosomal activity, and that the free phenol is not essential. This set of neolignan derivatives represents a promising starting point for future Chagas disease drug discovery studies.

Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and biological activity against Trypanosoma cruzi

Chagas disease is a neglected protozoan disease that affects more than eight million people in developing countries. Due to the limited number and toxicity profiles of therapies in current use, new drugs are urgently needed. In previous studies, we reported the isolation of two related antitrypanosomal neo- lignans from Nectandra leucantha (Lauraceae). In this work, a semi-synthetic library of twenty-three neolignan derivatives was prepared to explore synthetically accessible structure activity relationships (SAR) against Trypanosoma cruzi. Five compounds demonstrated activity against trypomastigotes (IC50 values from 8 to 64 mM) and eight showed activity against intracellular amastigotes (IC50 values from 7 to 16 mM). Eighteen derivatives demonstrated no mammalian cytotoxicity up to 200 mM. The phenolic ac- etate derivative of natural dehydrodieugenol

Pyrazinoates as antiparasitic agents against Trypanosoma cruzi.

This work reports a repurposing study of pyrazinoic acid (1) and methyl (2), ethyl (3) and 2-chloroethyl (4) ester derivatives with antimycobacterial activity, in assays against Trypanosoma cruzi. The compounds and benznidazole, the standard antitrypanosoma drug, were evaluated in concentrations ranging from 100 to 6.25 µg/mL. The results showed that compounds 2 and 3 (EC50 = 182 and 447 µM) significantly reduced the infection rate of the parasite into the mammalian cells at 100 µg/mL (p < 0.05) in a similar way to benznidazole. In addition, all the compounds also significantly reduced the number of intracellular parasites (compound 1 at 50 µg/mL, and compounds 2-4 at 100 µg/mL, p < 0.05) in comparison to the control. Compounds 1 and 2 were more effective than benznidazole at 50 µg/mL (p < 0.001). Moreover, compounds 1-4 did not show significant cytotoxicity against THP-1, J774, and HeLa cells (>1000 µM), indicating that they possess considerable selectivity against the parasites. This report represents the first study of such compounds against T. cruzi, indicating the potential of pyrazinoates as antiparasitic agents.

An overview on Leishmania (Mundinia) enriettii: biology, immunopathology, LRV and extracellular vesicles during the host-parasite interaction.

One of the Leishmania species known to be non-infective to humans is Leishmania (Mundinia) enriettii whose vertebrate host is the guinea pig Cavia porcellus. It is a good model for cutaneous leishmaniasis, chemotherapeutic and molecular studies. In the last years, an increased interest has emerged concerning the L. (Mundinia) subgenus after the finding of Leishmania (M.) macropodum in Australia and with the description of other new/putative species such as L. (M.) martiniquensis and 'L. (M.) siamensis'. This review focused on histopathology, glycoconjugates and innate immunity. The presence of Leishmania RNA virus and shedding of extracellular vesicles by the parasite were also evaluated.

Lipophosphoglycan polymorphisms do not affect Leishmania amazonensis development in the permissive vectors Lutzomyia migonei and Lutzomyia longipalpis.

BACKGROUND: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. RESULTS: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 ß-glucose or 1 to 3 ß-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. CONCLUSIONS: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

NLRP3 controls Trypanosoma cruzi infection through a caspase-1-dependent IL-1R-independent NO production.

Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1ß production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1ß secretion. NLRP3(-/-) and caspase1(-/-) mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88(-/-) and iNOS(-/-) mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3(-/-) and caspase1(-/-) mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1ß and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88(-/-) macrophages and rendered them as susceptible to T. cruzi infection as NLRP3(-/-) and caspase-1(-/-) macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.

Intraspecies variation in Trypanosoma cruzi GPI-mucins: biological activities and differential expression of α-galactosyl residues.

The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).

In vitro and in vivo trypanocidal effects of the cyclopalladated compound 7a, a drug candidate for treatment of Chagas' disease.

Chagas' disease, a neglected tropical infection, affects about 18 million people, and 100 million are at risk. The only drug available, benznidazole, is effective in the acute form and in the early chronic form, but its efficacy and tolerance are inversely related to the age of the patients. Side effects are frequent in elderly patients. The search for new drugs is thus warranted. In the present study we evaluated the in vitro and in vivo effect of a cyclopalladated compound (7a) against Trypanosoma cruzi, the agent of Chagas' disease. The 7a compound inhibits trypomastigote cell invasion, decreases intracellular amastigote proliferation, and is very effective as a trypanocidal drug in vivo, even at very low dosages. It was 340-fold more cytotoxic to parasites than to mammalian cells and was more effective than benznidazole in all in vitro and in vivo experiments. The 7a cyclopalladate complex exerts an apoptosis-like death in T. cruzi trypomastigote forms and causes mitochondrion disruption seen by electron microscopy.

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection.

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.

The surface coat of the mammal-dwelling infective trypomastigote stage of Trypanosoma cruzi is formed by highly diverse immunogenic mucins.

A thick coat of mucin-like glycoproteins covers the surface of Trypanosoma cruzi and plays a crucial role in parasite protection and infectivity and host immunomodulation. The appealing candidate genes coding for the mucins of the mammal-dwelling stages define a heterogeneous family termed TcMUC, which comprises up to 700 members, thus precluding a genetic approach to address the protein core identity. Here, we demonstrate by multiple approaches that the TcMUC II genes code for the majority of trypomastigote mucins. These molecules display a variable, non-repetitive, highly O-glycosylated central domain, followed by a short conserved C terminus and a glycosylphosphatidylinositol anchor. A simultaneous expression of multiple TcMUC II gene products was observed. Moreover, the C terminus of TcMUC II mucins, but not their central domain, elicited strong antibody responses in patients with Chagas' disease and T. crusi infected animals. This highly diverse coat of mucins may represent a refined parasite strategy to elude the mammalian host immune system.