ABSTRACT
Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.
Subject(s)
Extracellular Vesicles , Lipidomics , Lipids , Semen , Animals , Extracellular Vesicles/metabolism , Swine , Semen/metabolism , Semen/chemistry , Male , Lipids/analysis , Lipids/chemistry , Lipidomics/methods , Chromatography, High Pressure Liquid , Mass Spectrometry , Chromatography, GelABSTRACT
Fasciola hepatica and Dicrocoelium dendriticum are parasitic trematodes residing in the bile ducts of mammalian hosts, causing, in some cases, impairment of liver function and hepatic fibrosis. Previous studies have shown that extracellular vesicles released by F. hepatica (FhEVs) and D. dendriticum (DdEVs) induce a distinct phenotype in human macrophages, but there is limited information on the effect of parasitic EVs on liver cells, which interact directly with the worms in natural infections. In this study, we isolated FhEVs and DdEVs by size exclusion chromatography and labeled them with a lipophilic fluorescent dye to analyze their uptake by human hepatic stellate cells (HSC) and hepatocytes, important cell types in liver pathology, using synthetic liposomes as internal labeling and uptake control. We analyzed EV uptake and the proteome profiles after the treatment with EVs for both cell types. Our results reveal that EVs establish unique and specific interactions with stellate cells and hepatocytes, suggesting a different role of EVs derived from each parasite, depending on the migration route to reach their final niche. FhEVs have a cytostatic effect on HSCs, but induce the extracellular matrix secretion and elicit anti-inflammatory responses in hepatocytes. DdEVs have a more potent anti-proliferative effect than FhEVs and trigger a global inflammatory response, increasing the levels of NF-κB and other inflammatory mediators in both cell types. These interactions may have a major influence on the progression of the disease, serving to generate conditions that may favor the establishment of the helminths in the host.
ABSTRACT
The emergence of insecticide resistance in arbovirus vectors is putting the focus on the development of new strategies for control. In this regard, the exploitation of Wolbachia endosymbionts is receiving increasing attention due to its demonstrated effectiveness in reducing the vectorial capacity of Aedes mosquitoes. Here, we describe the establishment of a naïve Wolbachia infection in a wild Aedes albopictus population of eastern Spain through a hybridization approach to obtain males capable of sterilizing wild females. The obtained lines were compared with the Wolbachia donor, Ae. albopictus ARwP, previously artificially infected with Wolbachia wPip, regarding immature and adult survival, female fecundity, egg fertility, and level of induced sterility. Our results did not show significant differences between lines in any of the biological parameters analyzed, indicating the full suitability of the hybrids to be used as a control tool against Ae. albopictus. In particular, hybrid males induced 99.9% sterility in the eggs of wild females without the need for any preliminary treatment. Being harmless to non-target organisms and the environment, the use of this bacterium for the control of Ae. albopictus deserves further exploration. This is especially relevant in areas such as eastern Spain, where this mosquito species has recently spread and may represent a serious threat due to its competence as a vector for dengue, chikungunya, and Zika viruses.
ABSTRACT
Glioblastoma multiforme (GBM) is a malignancy of bad prognosis, and advances in early detection and treatment are needed. GBM is heterogenous, with varieties differing in malignancy within a tumor of a patient and between patients. Means are needed to distinguish these GMB forms, so that specific strategies can be deployed for patient management. We study the participation of the chaperone system (CS) in carcinogenesis. The CS is dynamic, with its members moving around the body in extracellular vesicles (EVs) and interacting with components of other physiological systems in health and disease, including GBM. Here, we describe the finding of high amounts of Hsp70 (HSPA1A) and the calcitonin receptor protein (CTR) in EVs in patients with GBM. We present a standardized protocol for collecting, purifying, and characterizing EVs carrying Hsp70 and CTR in plasma-derived EVs from patients with GBM. EVs from GBM patients were obtained just before tumor ablative surgery (T0) and 7 days afterwards (T1); Hsp70 was highly elevated at T0 and less so at T1, and CTR was greatly increased at T0 and reduced to below normal values at T1. Our results encourage further research to assess Hsp70 and CTR as biomarkers for differentiating tumor forms and to determine their roles in GBM carcinogenesis.
Subject(s)
Brain Neoplasms , Extracellular Vesicles , Glioblastoma , Humans , Glioblastoma/metabolism , Receptors, Calcitonin/metabolism , Cell Line, Tumor , Extracellular Vesicles/metabolism , Biomarkers, Tumor/metabolism , Carcinogenesis/metabolism , Brain Neoplasms/metabolismABSTRACT
Extracellular vesicles (EVs) are nanoparticles that are released by cells and participate in the transfer of information. It is now known that EVs from mammalian cells are involved in different physiological and pathophysiological processes (antigen presentation, tissue regeneration, cancer, inflammation, diabetes, etc.). In the past few years, several studies on plants have demonstrated that EVs are also key tools for plant intercellular and cross-kingdom communications, suggesting that these nanostructures may contribute to distinct aspects of plant physiology such as development, defense, reproduction, symbiotic relationships, etc. These findings are challenging the traditional view of signaling in plants. EVs are probably involved in the phloem's transport system, since this vascular tissue plays a crucial role in translocating nutrients, defensive compounds, and informational signals throughout the plant. The collection of phloem is experimentally challenging because sap is under high turgor pressure inside the sieve elements, which have a small diameter and are hidden within the plant organs. The goals of this work are to develop new protocols that allow us to detect EVs for the first time in the phloem of the plants, and to isolate these nanovesicles for in-depth analysis and characterization. Our protocols describe two distinct methods to collect the phloem sap from rice and melon. The first method (Basic Protocol 1) involves 'Aphid stylectomy by radiofrequency microcautery' using rice plants and the aphid Sitobion avenae. This is considered the least invasive method for collecting phloem sap. The second method, 'Stem incision', involves cutting the stem of melon plants for collecting the exuded sap. Phloem sap EVs are then isolated by size exclusion chromatography. The results obtained in this study represent the first report on typical EVs isolated from in vivo-collected phloem sap. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of EVs from phloem sap: Aphid stylectomy by radiofrequency microcautery Basic Protocol 2: Isolation of EVs from phloem sap: Stem incision method.
Subject(s)
Aphids , Extracellular Vesicles , Oryza , Animals , Phloem , Chromatography, Gel , Antigen Presentation , MammalsABSTRACT
Extracellular vesicles (EVs) released by the helminths Dicrocoelium dendriticum and Fasciola hepatica are important modulators of the host immune response, contributing to the establishment of the infection. Monocytes and, in particular, macrophages are major regulators of the inflammatory response and are likely responsible for the phagocytosis of most of the parasite EVs. In this study, we isolated EVs from F. hepatica (FhEVs) and D. dendriticum (DdEVs) by size exclusion chromatography (SEC) and characterized them by nanoparticle tracking analysis, transmission electron microscopy and LC-MS/MS, and analyzed the cohort of proteins. The treatment of monocytes/macrophages with FhEVs, DdEVs or EV-depleted fractions from SEC, demonstrated species-specific effects of the EVs. In particular, FhEVs reduce the migratory capacity of monocytes and the analysis of the cytokine profile showed that they induce a mixed M1/M2 response, exerting anti-inflammatory properties in Lipopolysaccharide-activated macrophages. In contrast, DdEVs do not affect monocyte migration and seem to have pro-inflammatory properties. These results correlate with the differences in the life cycle of both parasites, suggesting different host immune responses. Only F. hepatica migrates to the bile duct through the liver parenchyma, driving the host immune response to heal deep erosions. Furthermore, the proteomic analysis of the macrophages upon FhEV treatment identified several proteins that might be involved in FhEV-macrophage interactions.
Subject(s)
Dicrocoelium , Extracellular Vesicles , Fasciola hepatica , Animals , Humans , Fasciola hepatica/metabolism , Extracellular Vesicles/metabolism , Proteomics , Chromatography, Liquid , Tandem Mass Spectrometry , Macrophages/metabolismABSTRACT
There is increasing evidence that food is an important factor that influences the composition of the gut microbiota. Usually, all the attention has been focused on nutrients such as lipids, proteins, vitamins, or polyphenols. However, a pivotal role in these processes has been linked to dietary-derived exosome-like nanoparticles (DELNs). While food macro- and micronutrient composition are largely well established, there is considerable interest in these DELNs and their cargoes. In this sense, traditionally, all the attention was focused on the proteins or miRNAs contained in these vesicles. However, it has been shown that DELNs would also carry other bioactive molecules with a key role in regulating biochemical pathways and/or interactions with the host's gut microbiome affecting intracellular communication. Due to the scarce literature, it is necessary to compile the current knowledge about the antimicrobial capacity of DELNs and its possible molecular mechanisms that will serve as a starting point. For this reason, in this review, we highlight the impact of DENLs on different bacteria species modulating the host gut microbiota or antibacterial properties. It could be concluded that DELNs, isolated from both plant and animal foods, exert gut microbiota modulation. However, the presence of miRNA in the vesicle cargoes is not the only one responsible for this effect. Lipids present in the DELNs membrane or small molecules packed in may also be responsible for apoptosis signaling, inhibition, or growth promoters.
Subject(s)
Exosomes , MicroRNAs , Nanoparticles , Animals , MicroRNAs/metabolism , Bacteria/metabolism , Vitamins/metabolism , LipidsABSTRACT
OBJECTIVE: The presence of Aedes albopictus, of high sanitary and social impact, was first reported in Valencia (Eastern Spain) in 2015. Innovative tools for its control include the use of the endosymbiotic bacterium Wolbachia pipientis. The release of mosquito males infected with the wPip strain, has proven very promising for large-scale Incompatible Insect Technique (IIT) applications. Before this strategy can be implemented in Valencia, it is important to know whether the natural local mosquito populations are Wolbachia-infected and, if so, identifying the infecting strains/supergroups, these being the objectives of the present work. METHODS: Eggs were collected from the 19 districts of the València city between May and October 2019. A total of 50 lab-reared adult Ae. albopictus individuals were processed and analyzed for Wolbachia detection and molecular characterization. These actions took place within the framework of a collaboration established with the Department of Health and Consumer Affairs of the city council of Valencia. Fisher's exact test was used to detect the statistical significance of the differences between groups. RESULTS: Our study revealed that 94% of the analyzed samples were naturally infected with Wolbachia. Both wAlbA and wAlbB supergroups were identified, with most samples (72% of the infected ones) carrying co-infections. CONCLUSIONS: These data provide the first characterization of the Wolbachia presence in natural populations of Ae. albopictus in the Mediterranean area of Spain. This information is relevant to evaluate the potential use of Wolbachia strains in order to achieve the suppression of the Asian tiger mosquito populations through massive release of artificially-infected males.
OBJETIVO: La presencia de Aedes albopictus, de alto impacto sanitario y social, se informó por primera vez en Valencia en 2015. Las herramientas innovadoras para su control incluyen el uso de la bacteria endosimbiótica Wolbachia pipientis. La liberación de mosquitos machos infectados con la cepa wPip ha demostrado ser muy prometedora para aplicar la Técnica de Insectos Incompatibles (IIT) a gran escala. Antes de que esta estrategia pueda implementarse, es importante saber si las poblaciones locales de mosquitos silvestres están infectadas por Wolbachia y, de ser así, identificar las cepas/supergrupos infectantes, siendo estos los objetivos del presente trabajo. METODOS: Se recolectaron huevos de los diecinueve distritos de València entre mayo y octubre de 2019, y se mantuvieron en el laboratorio hasta llegar a adultos. Un total de cincuenta individuos adultos de Ae albopictus fueron procesados y analizados para detectar la presencia de Wolbachia y su caracterización molecular. Estas acciones se enmarcaron en la colaboración establecida con la Concejalía de Salud y Consumo del Ayuntamiento de València. La prueba exacta de Fisher fue utilizada para detectar la significación estadística de las diferencias entre grupos. RESULTADOS: El 94% de las muestras analizadas estaban infectadas de forma natural con Wolbachia. Se identificaron los supergrupos wAlbA y wAlbB, y la mayoría de las muestras (72% de las infectadas) presentaban coinfecciones. CONCLUSIONES: Los datos proporcionan la primera caracterización de la presencia de Wolbachia en poblaciones naturales de Ae. albopictus en el área mediterránea de España. Esta información es relevante para evaluar el potencial uso de cepas de Wolbachia de cara a la supresión de poblaciones de mosquito tigre asiático mediante la liberación masiva de machos infectados artificialmente.
Subject(s)
Aedes , Wolbachia , Adult , Male , Animals , Humans , Mosquito Control , Spain/epidemiologyABSTRACT
Fundamentos: La presencia deAedes albopictus, de alto impacto sanitario y social, se informó por primera vez en Valenciaen 2015. Las herramientas innovadoras para su control incluyen el uso de la bacteria endosimbióticaWolbachia pipientis. La liberación de mosquitos machos infectados con la cepawPip ha demostrado ser muy prometedora para aplicar la Técnica de InsectosIncompatibles (IIT) a gran escala. Antes de que esta estrategia pueda implementarse, es importante saber si las poblaciones localesde mosquitos silvestres están infectadas porWolbachia y, de ser así, identificar las cepas/supergrupos infectantes, siendo estos losobjetivos del presente trabajo. Métodos: Se recolectaron huevos de los diecinueve distritos de València entre mayo y octubre de 2019, y se mantuvieron en ellaboratorio hasta llegar a adultos. Un total de cincuenta individuos adultos deAe. albopictus fueron procesados y analizados paradetectar la presencia deWolbachia y su caracterización molecular. Estas acciones se enmarcaron en la colaboración establecida conla Concejalía de Salud y Consumo del Ayuntamiento de València. La prueba exacta de Fisher fue utilizada para detectar la significaciónestadística de las diferencias entre grupos. Resultados: El 94% de las muestras analizadas estaban infectadas de forma natural conWolbachia. Se identificaron los supergruposwAlbA ywAlbB, y la mayoría de las muestras (72% de las infectadas) presentaban coinfecciones. Conclusiones: Los datos proporcionan la primera caracterización de la presencia deWolbachia en poblaciones naturales deAe. albopictus en el área mediterránea de España. Esta información es relevante para evaluar el potencial uso de cepas deWolbachiade cara a la supresión de poblaciones de mosquito tigre asiático mediante la liberación masiva de machos infectados artificialmente.(AU)
Background: The presence ofAedes albopictus, of high sanitary and social impact, was first reported in Valencia (Eastern Spain)in 2015. Innovative tools for its control include the use of the endosymbiotic bacteriumWolbachia pipientis. The release of mosquitomales infected with thewPip strain, has proven very promising for large-scale Incompatible Insect Technique (IIT) applications. Beforethis strategy can be implemented in Valencia, it is important to know whether the natural local mosquito populations areWolbachia-infected and, if so, identifying the infecting strains/supergroups, these being the objectives of the present work. Methods: Eggs were collected from the 19 districts of the València city between May and October 2019. A total of 50 lab-reared adultAe. albopictus individuals were processed and analyzed forWolbachia detection and molecular characterization. These actions tookplace within the framework of a collaboration established with the Department of Health and Consumer Affairs of the city council ofValencia. Fishers exact test was used to detect the statistical significance of the differences between groups. Results: Our study revealed that 94% of the analyzed samples were naturally infected withWolbachia. BothwAlbA andwAlbBsupergroups were identified, with most samples (72% of the infected ones) carrying co-infections. Conclusions: These data provide the first characterization of theWolbachia presence in natural populations ofAe. albopictusin the Mediterranean area of Spain. This information is relevant to evaluate the potential use ofWolbachia strains in order to achievethe suppression of the Asian tiger mosquito populations through massive release of artificially-infected males.(AU)
Subject(s)
Humans , Mosquito Control , Culicidae , Wolbachia , Aedes , Eggs , Spain , Public Health , ResearchABSTRACT
Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.
Subject(s)
Extracellular Vesicles , Proteome , Male , Female , Animals , Swine , Proteome/metabolism , Semen/metabolism , Proteomics/methods , Extracellular Vesicles/metabolism , Mass SpectrometryABSTRACT
Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.
Subject(s)
Extracellular Vesicles , Helminths , Animals , Humans , Extracellular Vesicles/physiology , Reproducibility of Results , MammalsABSTRACT
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.
ABSTRACT
Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.
Subject(s)
Chagas Disease , Exosomes , Extracellular Vesicles , Trypanosoma cruzi , Animals , Cell Communication , Chagas Disease/therapyABSTRACT
In the current study, extracellular vesicles from pomegranate juice (PgEVs) were isolated for the first time using size exclusion chromatography (SEC). This method permitted us to obtain highly enriched EV samples without most of the non-EV co-isolated proteins. The characterization of PgEVs through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) allowed the determination of vesicles' concentration/volume, size, and morphology. It was confirmed from the analytical data that PgEVs contain a homogeneous population of vesicles, with a dimension and structure comparable to plant-derived EVs. Proteomic analyses by LC-MS/MS led to the characterization of 131 proteins, and several of them were related commonly to the biogenesis and transport of EVs, and/or proposed as EV markers. PgEVs exerted anti-inflammatory, antioxidant and wound-healing effects when added to the in vitro cultures of monocytic (THP-1) and intestinal (Caco-2) cell lines, respectively.
Subject(s)
Extracellular Vesicles , Proteomics , Humans , Proteomics/methods , Chromatography, Liquid , Caco-2 Cells , Tandem Mass Spectrometry , Extracellular Vesicles/metabolism , Proteins/metabolismABSTRACT
Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder, characterized by the misfolding and aggregation of α-synuclein (α-syn) into Lewy bodies and the degeneration of dopaminergic neurons in the substantia nigra pars compacta. The urge for an early diagnosis biomarker comes from the fact that clinical manifestations of PD are estimated to appear once the substantia nigra has deteriorated and there has been a reduction of the dopamine levels from the striatum. Nowadays, extracellular vesicles (EVs) play an important role in the pathogenesis of neuro-degenerative diseases as PD. A systematic review dated August 2022 was carried out with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses with the aim to analyze the potential role of EVs as biomarkers for PD. From a total of 610 articles retrieved, 29 were eligible. This review discusses the role of EVs biochemistry and their cargo proteins, such as α-syn and DJ-1 among others, detected by a proteomic analysis as well as miRNAs and lncRNAs, as potential biomarkers that can be used to create standardized protocols for early PD diagnosis as well as to evaluate disease severity and progression.
Subject(s)
Extracellular Vesicles , MicroRNAs , Parkinson Disease , RNA, Long Noncoding , Biomarkers/metabolism , Dopamine , Dopaminergic Neurons/metabolism , Extracellular Vesicles/metabolism , Humans , Parkinson Disease/metabolism , Proteomics , alpha-Synuclein/metabolismABSTRACT
Douanne et al. recently reported horizontal gene transfer (HGT) mediated by extracellular vesicles (EVs) in Leishmania; this constitutes the first report of this phenomenon in parasites. They showed that EVs facilitate the transmission of drug-resistance genes and increased cell fitness in stressful environments, indicating potential clinical application of these findings.
Subject(s)
Extracellular Vesicles , Leishmania , Parasites , Animals , Drug Resistance , Gene Transfer, Horizontal , Leishmania/geneticsABSTRACT
Strongyloidiasis, caused by Strongyloides stercoralis, is a neglected parasitic disease that represents a serious public health problem. In immunocompromised patients, this parasitosis can result in hyperinfection or disseminated disease with high levels of mortality. In previous studies, the mRNAs encoding for the 14-3-3 and major antigen proteins were found to be expressed at high levels in S. stercoralis L3 larvae, suggesting potential key roles in parasite-host interactions. We have produced them as recombinant proteins (rSs14-3-3 and rSsMA) in a bacterial protein expression system. The serum levels of anti-rSs14-3-3 and anti-rSsMA IgGs are increased upon infection with S. venezuelensis, validating the use of the mouse model since the native 14-3-3 and MA proteins induce an immune response. Each recombinant protein was formulated in the adjuvant adaptation (ADAD) vaccination system and injected twice, subcutaneously, in CD1 mice that were experimentally infected with 3000 S. venezuelensis L3 to evaluate their protective and immunomodulatory activity. Our results, including the number of parthenogenetic females, number of eggs in stool samples and the analysis of the splenic and intestinal indexes, show that the vaccines did not protect against infection. The immunization with rSs14-3-3 induced changes in the cytokine profile in mice, producing higher expression of IL-10, TGF-ß, IL-13 and TNF-α in the spleen, suggesting a Th2/Treg-type response with an increase in TNF-α levels, confirming its role as an immunomodulator.
ABSTRACT
The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.
Subject(s)
Extracellular Vesicles , Fasciola hepatica , Animals , Chromatography, Liquid , Extracellular Vesicles/metabolism , Fasciola hepatica/chemistry , Fasciola hepatica/metabolism , Helminth Proteins/metabolism , Proteomics , Tandem Mass SpectrometryABSTRACT
Extracellular vesicles (EVs) have been described in all eukaryotic and prokaryotic cells as released membranous structures loaded with biomolecules including nucleic acids, glycoconjugates, lipids and proteins. Two main groups of vesicles with different biogenesis and size are considered to be the most predominant, Exosomes (30-100 nm) originating from multivesicular bodies, and microvesículas (100-1000 nm) originating from plasma membrane. EVs participate in cellular communication between different organisms and can alter neighbour cells, participating in physiological and pathophysiological processes. In this issue, eleven reviews summarize the current knowledge in the characterization of EVs participating in the pathogenic-host interaction including protozoa, helminths, bacteria, fungi and viruses (Montaño et al., 2021; Palacios et al., 2021; Rossi et al., 2021; Sabatke et al., 2021; Cucher et al., 2021; Gilmore W et al., 2021; Sánchez-López et al., 2021; Dong et al., 2021; Drurey C and Mayzels R.M., 2021; Macedo-Da Silva J et al., 2021; Piffer, A. C et al., 2021).
Subject(s)
Cell Communication/immunology , Extracellular Vesicles/immunology , Host-Pathogen Interactions/immunology , Infections/immunology , Animals , HumansABSTRACT
BACKGROUND: Because of their specific and biologically relevant cargo, urine extracellular vesicles (EVs) constitute a valuable source of potential non-invasive biomarkers that could support the clinical decision-making to improve the management of prostate cancer (PCa) patients. Different EV isolation methods differ in terms of complexity and yield, conditioning, as consequence, the analytical result. METHODS: The aim of this study was to compare three different isolation methods for urine EVs: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial kit (Exolute® Urine Kit). Urine samples were collected from 6 PCa patients and 4 healthy donors. After filtered through 0.22 µm filters, urine was divided in 3 equal volumes to perform EVs isolation with each of the three approaches. Isolated EVs were characterized by spectrophotometric protein quantification, nanoparticle tracking analysis, transmission electron microscopy, AlphaScreen Technology, and whole miRNA Transcriptome. RESULTS: Our results showed that UC and SEC provided better results in terms of EVs yield and purity than Exolute®, non-significant differences were observed in terms of EV-size. Interestingly, luminescent AlphaScreen assay demonstrated a significant enrichment of CD9 and CD63 positive microvesicles in SEC and UC methods compared with Exolute®. This heterogeneity was also demonstrated in terms of miRNA content indicating that the best correlation was observed between UC and SEC. CONCLUSIONS: Our study highlights the importance of standardizing the urine EV isolation methods to guaranty the analytical reproducibility necessary for their implementation in a clinical setting.