Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis

ABSTRACT Purpose: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. Methods: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. Results: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. Conclusion: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

Extracellular vesicles in malaria: Pathogenesis, diagnosis and therapy.

Malaria is a life-threatening disease caused by parasites from the genus Plasmodium. Five species can cause malaria in humans, with Plasmodium vivax being the most common in many countries and Plasmodium falciparum having the highest lethality, which can lead to cerebral malaria. Extracellular vesicles (EVs) are in focus in malaria research to better understand pathogenesis, diagnosis, therapy, and prognosis. Malaria-causing parasites use EVs to transfer their molecules to host cells, a mechanism that significantly contributes to parasite survival and successful infection. EVs have thus emerged as an essential component of the immunopathological cascade of malaria, playing a pivotal role in disease progression and severity. This chapter discusses the epidemiology and pathogenesis of malaria and the role of EVs as new diagnostic and therapeutic tools, emphasizing their potential clinical significance.

Involvement of extracellular vesicles in the interaction of hosts and Toxoplasma gondii.

Toxoplasma gondii, the causative agent of toxoplasmosis, is widely distributed. This protozoan parasite is one of the best adapted, being able to infect innumerous species of animals and different types of cells. This chapter reviews current literature on extracellular vesicles secreted by T. gondii and by its hosts. The topics describe the life cycle and transmission (1); toxoplasmosis epidemiology (2); laboratorial diagnosis approach (3); The T. gondii interaction with extracellular vesicles and miRNAs (4); and the perspectives on T. gondii infection. Each topic emphases the host immune responses to the parasite antigens and the interaction with the extracellular vesicles and miRNAs.

Extracellular vesicles: Methods for purification and characterization.

Extracellular vesicles (EVs) are membrane-bound particles released by cells that play a significant role in intercellular communication. They can be obtained from a variety of sources, including conditioned culture medium, blood and urine. In this chapter we detail the methods for EV isolation and characterization. Isolating and characterizing EVs is essential for understanding their functions in physiological and pathological processes. Advances in isolation and characterization techniques provide opportunities for deeper research into EV biology and its potential applications in diagnostics and therapeutics.

Extracellular vesicles release from protozoa parasite and animal model.

Diseases caused by protozoan parasites, such as leishmaniasis, trypanosomiasis, and malaria, are highly complex and together continue to cause high annual morbidity and mortality. The search for new compounds in environmental biodiversity, repositioning known drugs, and developing vaccines using old and innovative technologies have been employed to discover vaccines and new and alternative treatments. Extracellular vesicles (EVs) can carry parasite antigens, creating a new possibility to develop an effective and affordable platform for treatment, vaccines, and drug delivery. Thus, the evaluation of EVs in animal models can and should be explored among the countless biomedical applications. Herein, we will address the concept of EVs, their acquisition and characterization in protozoan parasite models, and the primary studies using these vesicles in therapeutic applications.

Biogenesis of EVs in Trypanosomatids.

Trypanosomes are protozoan parasites responsible for human diseases such as Chagas disease, African trypanosomiasis, and leishmaniasis. These organisms' growth in various environments and exhibit multiple morphological stages, while adapting their surface components. They acquire and release materials extensively to get nutrients and manage interactions with the extracellular environment. They acquire and utilize proteins, lipids, and carbohydrates for growth via using membrane transport and endocytosis. Endocytosis takes place through distinct membrane areas known as the flagellar pocket and cytostome, depending on the parasite species and its developmental stage. Some forms establish a complex endocytic system to either store or break down the absorbed materials. In contrast, membrane transport facilitates the uptake of small molecules like amino acids, carbohydrates, and iron via particular receptors on the plasma membrane. Concurrently, these parasites secrete various molecules such as proteins, enzymes, nucleic acids, and glycoconjugates either in soluble form or enclosed in extracellular vesicles, which significantly contribute to their parasitic behavior. These activities require exocytosis through a secretory pathway in certain membrane domains such as the flagellum, flagellar pocket, and plasma membrane, which are controlled at various developmental stages. The main features of the endocytic and exocytic mechanisms, as well as the organelles involved, are discussed in this chapter along with their connection to the formation of exosomes and extracellular vesicles in the Tritryp species.

Extracellular vesicles.

Cells, pathogens, and other systems release extracellular vesicles (EVs). The particles promote intercellular communication and contain proteins, lipids, RNA and DNA. Initially considered to be cellular waste in the twentieth century, EVs were becoming recognized for their function in biological communication and control. EVs are divided into many subtypes: exosomes, microvesicles, and apoptotic bodies. Exosomes form in the late endosome/multivesicular body and are released when the compartments fuse with the plasma membrane. Microvesicles are generated by direct budding of the plasma membrane, whereas apoptotic bodies are formed after cellular apoptosis. The new guideline for EVs that describes alternate nomenclature for EVs. The particles modulate the immune response by affecting both innate and adaptive immunity, and their specific the structure allows them to be used as biomarkers to diagnose a variety of diseases. EVs have a wide range of applications, for example, delivery systems for medications and genetic therapies because of their ability to convey specific cellular material. In anti-tumor therapy, EVs deliver therapeutic chemicals to tumor cells. The EVs promote transplant compatibility and reduce organ rejection. Host-parasite interactions, therapeutic and diagnostic for cancer, cardiovascular disease, cardiac tissue regeneration, and the treatment of neurological diseases such as Alzheimer's and Parkinson's. The study of EVs keeps on expanding, revealing new functions and beneficial options. EVs have the potential to change drug delivery, diagnostics, and specific therapeutics, creating a new frontier in biomedical.

Contribution of extracellular vesicles to steatosis-related liver disease and their therapeutic potential.

Extracellular vesicles (EVs) are small particles released by many cell types in different tissues, including the liver, and transfer specific cargo molecules from originating cells to receptor cells. This process generally culminates in activation of distant cells and inflammation and progression of certain diseases. The global chronic liver disease (CLD) epidemic is estimated at 1.5 billion patients worldwide. Cirrhosis and liver cancer are the most common risk factors for CLD. However, hepatitis C and B virus infection and obesity are also highly associated with CLD. Nonetheless, the etiology of many CLD pathophysiological, cellular, and molecular events are unclear. Changes in hepatic lipid metabolism can lead to lipotoxicity events that induce EV release. Here, we aimed to present an overview of EV features, from definition to types and biogenesis, with particular focus on the molecules related to steatosis-related liver disease, diagnosis, and therapy.

Small extracellular vesicles derived from the crosstalk between early embryos and the endometrium potentially mediate corpus luteum function.

The first interactions among the embryo, endometrium, and corpus luteum (CL) are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that sEVs from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: 1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; 2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release sEVs with different miRNA contents, and; 3) the luteal explant (CLE) exposed to these sEVs have distinct mRNA and miRNA profiles. To elucidate this, the EE were cultured in the presence or absence of a single Day-7 in vivo (EE-AI) or in vitro (EE-IVF) embryo. After of culture we found, in the EE, 45 and 211 differentially expressed genes (DEGs) associated with embryo presence and origin, respectively. SEVs were recovered from the conditioned media (CM) in which EE and embryos were co-cultured. Four miRNAs were differentially expressed between sEVs from CM-EE-AI and CM-EE-IVF. Luteal explants exposed in culture to these sEVs showed 1360 transcripts, and fifteen miRNAs differentially expressed. The DEGs associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release sEVs capable of modifying the mRNA and miRNA profile in the CL. Therefore, the sEVs-mediated embryo-endometrium-CL interactions possibly regulate the CL viability to ensure pregnancy success.

Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles as Natural Nanocarriers in the Treatment of Nephrotoxic Injury In Vitro.

Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UC-EVs) are valuable in nanomedicine as natural nanocarriers, carrying information molecules from their parent cells and fusing with targeted cells. miRNA-126, specific to endothelial cells and derived from these vesicles, supports vascular integrity and angiogenesis and has protective effects in kidney diseases. OBJECTIVE: This study investigates the delivery of miRNA-126 and anti-miRNA-126 via UC-EVs as natural nanocarriers for treating nephrotoxic injury in vitro. METHOD: The umbilical cord-derived mesenchymal stem cell and UC-EVs were characterized according to specific guidelines. Rat kidney proximal tubular epithelial cells (tubular cells) were exposed to nephrotoxic injury through of gentamicin and simultaneously treated with UC-EVs carrying miRNA-126 or anti-miRNA-126. Specific molecules that manage cell cycle progression, proliferation cell assays, and newly synthesized DNA and DNA damage markers were evaluated. RESULTS: We observed significant increases in the expression of cell cycle markers, including PCNA, p53, and p21, indicating a positive cell cycle regulation with newly synthesized DNA via BrDU. The treatments reduced the expression of DNA damage marker, such as H2Ax, suggesting a lower rate of cellular damage. CONCLUSIONS: The UC-EVs, acting as natural nanocarriers of miRNA-126 and anti-miRNA-126, offer nephroprotective effects in vitro. Additionally, other components in UC-EVs, such as proteins, lipids, and various RNAs, might also contribute to these effects.

Exploring cell-derived extracellular vesicles in peripheral blood and bone marrow of B-cell acute lymphoblastic leukemia pediatric patients: proof-of-concept study.

Extracellular vesicles (EVs) are heterogeneous, phospholipid membrane enclosed particles that are secreted by healthy and cancerous cells. EVs are present in diverse biological fluids and have been associated with the severity of diseases, which indicates their potential as biomarkers for diagnosis, prognosis and as therapeutic targets. This study investigated the phenotypic characteristics of EVs derived from peripheral blood (PB) and bone marrow (BM) in pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) during different treatment stages. PB and BM plasma were collected from 20 B-ALL patients at three time points during induction therapy, referred to as: diagnosis baseline (D0), day 15 of induction therapy (D15) and the end of the induction therapy (D35). In addition, PB samples were collected from 10 healthy children at a single time point. The EVs were measured using CytoFLEX S flow cytometer. Calibration beads were employed to ensure accurate size analysis. The following, fluorescent-labeled specific cellular markers were used to label the EVs: Annexin V (phosphatidylserine), CD235a (erythrocyte), CD41a (platelet), CD51 (endothelial cell), CD45 (leukocyte), CD66b (neutrophil), CD14 (monocyte), CD3 (T lymphocyte), CD19, CD34 and CD10 (B lymphoblast/leukemic blast). Our results demonstrate that B-ALL patients had a marked production of EV-CD51/61+, EV-CD10+, EV-CD19+ and EV-CD10+CD19+ (double-positive) with a decrease in EV-CD41a+ on D0. However, the kinetics and signature of production during induction therapy revealed a clear decline in EV-CD10+ and EV-CD19+, with an increase of EV-CD41a+ on D35. Furthermore, B-ALL patients showed a complex biological network, exhibiting distinct profiles on D0 and D35. Interestingly, fold change and ROC curve analysis demonstrated that EV-CD10+CD19+ were associated with B-ALL patients, exhibited excellent clinical performance and standing out as a potential diagnostic biomarker. In conclusion, our data indicate that EVs represent a promising field of investigation in B-ALL, offering the possibility of identifying potential biomarkers and therapeutic targets.

Changes in the Release of Endothelial Extracellular Vesicles CD144+, CCR6+, and CXCR3+ in Individuals with Acute Myocardial Infarction.

Acute myocardial infarction (AMI) results from vulnerable plaque rupture, causing ischemic cardiomyocyte necrosis and intense inflammation. Paradoxically, this inflammation releases factors that aid heart repair. Recent findings suggest a role for extracellular vesicles (EVs) in intercellular communication during post-AMI cardiac repair. However, EVs' tissue origin and chemokine profile in the blood of patients with AMI remains unclear. This study characterized the tissue origin and chemokine receptor profile of EVs in the coronary and peripheral blood of patients with AMI. The results reveal that vesicles isolated from coronary and peripheral blood plasma are enriched in tetraspanin (CD9) and express CD81+, CD90+, and CD144+. The vesicle size ranged between 145 and 162 nm, with the control group exhibiting smaller vesicles (D10) than the AMI group. Furthermore, all vesicles expressed CCR6 and CXCR3, whereas a small percentage expressed CCR4. In addition, a decrease in CXCR3 and CCR6 expression was observed in coronary and peripheral AMI blood vesicles compared with the control; however, no difference was found between AMI coronary and AMI peripheral blood vesicles. In conclusion, our study demonstrates, for the first time, changes in the number of extracellular vesicles expressing CD144+, CXCR3, and CCR6 in the peripheral circulation of patients with AMI. Extracellular vesicles present in the circulation of patients with AMI hold excellent promise as a potential diagnostic tool.

Extracellular vesicles: emerging paradigms in bovine embryo-maternal communication.

The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.

Cytokine secretion by in vitro cultures of lung epithelial cells, differentiated macrophages and differentiated dendritic cells incubated with pneumococci and pneumococcal extracellular vesicles.

Streptococcus pneumoniae is an important human pathogen that can colonize the respiratory tract of healthy individuals. The respiratory tract mucosa is thus the first barrier for this pathogen. In this study, we have tested three models of the respiratory epithelium with immune cells: (i) monolayer of A549 human lung epithelial cells, (ii) A549 + macrophages differentiated from the human monocytic THP-1 cell line (dMφ) and (iii) A549 + dMφ + dendritic cells differentiated from THP-1 (dDC) using a two-chamber system. Pneumococcal strains Rx1 (non-encapsulated) and BHN418 (serotype 6B) were incubated with the cells and secretion of IL-6, IL-8, IL-1ß, TNF-α and IL-10 was evaluated. Overall, the models using co-cultures of A549 + dMφ and A549 + dMφ + dDC elicited higher levels of pro-inflammatory cytokines and the non-encapsulated strain elicited an earlier cytokine response. BHN418 pspA (pneumococcal surface protein A) and pspC (pneumococcal surface protein C) knockouts elicited similar cytokine secretion in the co-culture models, whereas BHN18 ply (pneumolysin) knockout induced much lower levels. The results are in accordance with the activation of the inflammasome by Ply. Finally, we evaluated pneumococcal extracellular vesicles (pEVs) in the co-culture models and observed secretion of pro-inflammatory cytokines in the absence of cytotoxicity. Since pEVs are being studied as vaccine candidate against pneumococcal infections, the co-cultures of A549 + dMφ and A549 + dMφ + dDC are simple models that could be used to evaluate pEV vaccine batches.

Extracellular vesicles released by Trypanosoma evansi: induction analysis and proteomics.

Trypanosoma evansi is a unicellular protozoan responsible for causing a disease known as "surra," which is found in different regions of the world and primarily affects horses and camels. Few information is known about virulence factors released from the parasite within the animals. The organism can secrete extracellular vesicles (EVs), which transport a variety of molecules, including proteins. Before being considered exclusively as a means for eliminating unwanted substances, extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating interactions between cells, host cells, and parasites, and even between parasites themselves. Thus, they may be used as potential biomarkers. This study aimed to assess the induction of EVs production by Ca+2, conduct a proteomic analysis of the EVs released by T. evansi, and identify epitopes that could serve as biomarkers. The findings indicated that Ca+2 is not an effective promoter of vesiculation in T. evansi. Furthermore, the proteomic analysis has identified multiple proteins that have been investigated as biomarkers or vaccine antigens, previously. A total of 442 proteins were identified, with 7 of them specifically recognizing 9 epitopes that are unique to T. evansi. At least one of these epitopes of TevSTIB805.9.11580 have been previously identified, which increases the possibility of further investigating its potential as a biomarker.

Corpus luteum proximity alters molecular signature of the small extracellular vesicles and cumulus cells in the bovine ovarian follicle environment.

Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells (CC) and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that follicles from the ovary ipsilateral to the corpus luteum (CL) containing high intrafollicular P4 concentrations enhance the abundance of proteins detected in follicular-derived small extracellular vesicles (sEVs) predicted to be involved in the retraction of membrane projections based on actin filaments, such as transzonal projections (TZPs). Conversely, we found that follicles from the ovary contralateral to the CL, which contained low intrafollicular P4 concentrations, had a high detection of proteins predicted to regulate the maintenance of TZPs. We also performed RNAseq analysis which demonstrated that 177 genes were differentially expressed in CC under the different P4 environments. Bioinformatic analysis points to changes associated to cell metabolism in cells from follicles ipsilateral to the CL in comparison to genes involved in cell communication in CC from follicles contralateral to the CL. Our functional analysis experiment confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence.

Oligodendrocyte precursor cell-derived exosomes combined with cell therapy promote clinical recovery by immunomodulation and gliosis attenuation.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by autoimmune destruction of the myelin sheath, leading to irreversible and progressive functional deficits in patients. Pre-clinical studies involving the use of neural stem cells (NSCs) have already demonstrated their potential in neuronal regeneration and remyelination. However, the exclusive application of cell therapy has not proved sufficient to achieve satisfactory therapeutic levels. Recognizing these limitations, there is a need to combine cell therapy with other adjuvant protocols. In this context, extracellular vesicles (EVs) can contribute to intercellular communication, stimulating the production of proteins and lipids associated with remyelination and providing trophic support to axons. This study aimed to evaluate the therapeutic efficacy of the combination of NSCs and EVs derived from oligodendrocyte precursor cells (OPCs) in an animal model of multiple sclerosis. OPCs were differentiated from NSCs and had their identity confirmed by gene expression analysis and immunocytochemistry. Exosomes were isolated by differential ultracentrifugation and characterized by Western, transmission electron microscopy and nanoparticle tracking analysis. Experimental therapy of C57BL/6 mice induced with experimental autoimmune encephalomyelitis (EAE) were grouped in control, treated with NSCs, treated with OPC-derived EVs and treated with a combination of both. The treatments were evaluated clinically using scores and body weight, microscopically using immunohistochemistry and immunological profile by flow cytometry. The animals showed significant clinical improvement and weight gain with the treatments. However, only the treatments involving EVs led to immune modulation, changing the profile from Th1 to Th2 lymphocytes. Fifteen days after treatment revealed a reduction in reactive microgliosis and astrogliosis in the groups treated with EVs. However, there was no reduction in demyelination. The results indicate the potential therapeutic use of OPC-derived EVs to attenuate inflammation and promote recovery in EAE, especially when combined with cell therapy.

EV-miRNAs from breast cancer patients of plasma as potential prognostic biomarkers of disease recurrence.

Background: Extracellular vesicles (EVs), ubiquitously released by blood cells, facilitate intercellular communication. In cancer, tumor-derived EVs profoundly affect the microenvironment, promoting tumor progression and raising the risk of recurrence. These EVs contain miRNAs (EV-miRNAs), promising cancer biomarkers. Characterizing plasma EVs and identifying EV-miRNAs associated with breast cancer recurrence are crucial aspects of cancer research since they allow us to discover new biomarkers that are effective for understanding tumor biology and for being used for early detection, disease monitoring, or approaches to personalized medicine. This study aimed to characterize plasma EVs in breast cancer (BC) patients and identify EV-miRNAs associated with BC recurrence. Methods: This retrospective observational study included 24 BC patients divided into recurrence (n= 11) and non-recurrence (n= 13) groups. Plasma EVs were isolated and characterized. Total RNA from EVs was analyzed for miRNA expression using NanoString's nCounter® miRNA Expression Assays panel. MicroRNA target prediction used mirDIP, and pathway interactions were assessed via Reactome. Results: A stronger presence of circulating EVs was found to be linked with a less favorable prognosis (p = 0.0062). We discovered a distinct signature of EV-miRNAs, notably including miR-19a-3p and miR-130b-3p, which are significantly associated with breast cancer recurrence. Furthermore, miR-19a-3p and miR-130b-3p were implicated in the regulation of PTEN and MDM4, potentially contributing to breast cancer progression.A notable association emerged, indicating a high concentration of circulating EVs predicts poor prognosis (p = 0.0062). Our study found a distinct EV-miRNA signature involving miR-19a-3p and miR-130b-3p, strongly associated with disease recurrence. We also presented compelling evidence for their regulatory roles in PTEN and MDM4 genes, contributing to BC development. Conclusion: This study revealed that increased plasma EV concentration is associated with BC recurrence. The prognostic significance of EVs is closely tied to the unique expression profiles of miR-19a-3p and miR-130b-3p. These findings underscore the potential of EV-associated miRNAs as valuable indicators for BC recurrence, opening new avenues for diagnosis and treatment exploration.

Potential of extracellular vesicles in the pathogenesis, diagnosis and therapy for parasitic diseases.

Parasitic diseases have a significant impact on human and animal health, representing a major hazard to the public and causing economic and health damage worldwide. Extracellular vesicles (EVs) have long been recognized as diagnostic and therapeutic tools but are now also known to be implicated in the natural history of parasitic diseases and host immune response modulation. Studies have shown that EVs play a role in parasitic disease development by interacting with parasites and communicating with other types of cells. This review highlights the most recent research on EVs and their role in several aspects of parasite-host interactions in five key parasitic diseases: Chagas disease, malaria, toxoplasmosis, leishmaniasis and helminthiases. We also discuss the potential use of EVs as diagnostic tools or treatment options for these infectious diseases.