Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells

The extracellular vesicles (EVs) released by Leishmania can contribute to the establishment of infection and host immunomodulation. In this study, we characterized the shedding of EVs from Leishmania (Leishmania) amazonensis promastigotes. This species is the causative agent of cutaneous leishmaniasis, and its role during interactions with bone marrow-derived macrophages (BMDMs) and peritoneal B-1 cells was evaluated. Leishmania amazonensis promastigotes cultivated in vitro at different times and temperatures spontaneously released EVs. EVs were purified using size-exclusion chromatography (SEC) and quantitated by nanoparticle tracking analysis (NTA). NTA revealed that the average size of the EVs was approximately 180 nm, with concentrations ranging from 1.8 × 108 to 2.4 × 109 vesicles/mL. In addition, the presence of LPG and GP63 were detected in EVs obtained at different temperatures. Naïve BMDMs stimulated with EVs exhibited increased IL-10 and IL-6 expression. However, incubating B-1 cells with parasite EVs did not stimulate IL-10 expression but led to an increase in the expression of IL-6 and TNFα. After 7 weeks post-infection, animals infected with L. amazonensis promastigotes in the presence of parasite EVs had significant higher parasite load and a polarization to Th2 response, as compared to the group infected with the parasite alone. This work demonstrated that EVs isolated from L. amazonensis promastigotes were able to stimulate macrophages and B-1 cells to express different types of cytokines. Moreover, the immunomodulatory properties of EVs probably contributed to an increase in parasite burden in mice. These findings suggest that the functionality of L. amazonensis EVs on immune system favor of parasite survival and disease progression

Large-scale production of extracellular vesicles: Report on the “massivEVs” ISEV workshop

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor-made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field

Extracellular Vesicles: Role in Inflammatory Responses and Potential Uses in Vaccination in Cancer and Infectious Diseases

Submitted by Nuzia Santos ([email protected]) on 2016-03-07T13:27:23Z No. of bitstreams: 1 Extracellular Vesicles.pdf: 769134 bytes, checksum: a351cbec1a2646a11161b6f8ba81481a (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-03-07T14:03:47Z (GMT) No. of bitstreams: 1 Extracellular Vesicles.pdf: 769134 bytes, checksum: a351cbec1a2646a11161b6f8ba81481a (MD5)Made available in DSpace on 2016-03-07T14:03:47Z (GMT). No. of bitstreams: 1 Extracellular Vesicles.pdf: 769134 bytes, checksum: a351cbec1a2646a11161b6f8ba81481a (MD5) Previous issue date: 2015Universidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brasil.Universidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, Brasil.Almost all cells and organisms release membrane structures containing proteins, lipids, and nucleic acids called extracellular vesicles (EVs), which have a wide range of functions concerning intercellular communication and signaling events. Recently, the characterization and understanding of their biological role have become a main research area due to their potential role in vaccination, as biomarkers antigens, early diagnostic tools, and therapeutic applications. Here, we will overview the recent advances and studies of Evs shed by tumor cells, bacteria, parasites, and fungi, focusing on their inflammatory role and their potential use in vaccination and diagnostic of cancer and infectious diseases

Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics

Leishmania braziliensis and Leishmania amazonensis amastigote extracts differ in their enhancement effect on Leishmania infection when injected intradermally.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-08-22T18:16:44Z No. of bitstreams: 1 Araujo C.F Leishmania Braziliensis and ......pdf: 765622 bytes, checksum: 7059aebf2ad3bf5379b831d1aaf123b5 (MD5)Made available in DSpace on 2014-08-22T18:16:44Z (GMT). No. of bitstreams: 1 Araujo C.F Leishmania Braziliensis and ......pdf: 765622 bytes, checksum: 7059aebf2ad3bf5379b831d1aaf123b5 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilState University of Southwest Bahia. Jequié, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilBACKGROUND: It has been reported that repeated intravenous injections of a relatively large amount of Leishmania amazonensis amastigote extract (LaE) in BALB/c mice exacerbates the infection of these mice by Leishmania braziliensis. The identification of the extract active principle(s) through physicochemical purification often involves dilution and losses of protein in the course of successive purification procedures. The large amount of the extract required to induce the phenomenon, therefore, hinders the carrying out of experiments aimed at identifying the active molecule(s) through extract purification. In the present work, a dose-response experiment was done to find out if smaller amounts of LaE than that necessary to be used by the intravenous route would reproduce the phenomenon when injected by the intradermal route. In addition, it was also investigated whether a Leishmania braziliensis amastigote extract (LbE) would exert the same effect and whether the effect would occur in C57BL/6 mice. RESULTS: It was found that a single injection of either LaE or LbE containing 5 µg of protein was capable of enhancing the infection in BALB/c but not in C57BL/6 mice. In addition, it was observed that the largest tested doses of LbE (containing 30 and 180 µg of protein) failed to enhance the infection by L. braziliensis, whereas all doses of LaE enhanced equally that infection. CONCLUSIONS: Those results indicate the possible existence in LbE, and not in LaE, of molecules that interfere with the extract infection-enhancing activity when it is injected in large amounts, and that the inoculation of Leishmania extracts through the intravenous and intradermal routes potentiate the infection by L. braziliensis through the same mechanism

Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions

Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies

Extracellular Vesicles From Sporothrix brasiliensis Are an Important Virulence Factor That Induce an Increase in Fungal Burden in Experimental Sporotrichosis

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-α cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection