Agarose spot migration assay to measure the chemoattractant potential of extracellular vesicles: applications in regenerative medicine and cancer metastasis.

BACKGROUND: The recruitment of effector cells is one of the novel functions described for extracellular vesicles (EVs) that needs further study. For instance, cell recruitment by mesenchymal stromal cell derived-EVs (MSC-EVs) is one of the features by which MSC-EVs may induce regeneration and ameliorate tissue injury. On the other hand, increasing evidence suggests that cancer EVs play an important role in the preparation of the pre-metastatic niche (PMN) by recruiting their primary tumour cells. Understanding and measuring the potential of MSC-EVs or cancer-EVs to induce cell migration and recruitment is essential for cell-free therapeutic approaches and/or for a better knowledge of cancer metastasis, respectively. In this context, classical in vitro migration assays do not completely mimic the potential situation by which EVs exert their chemotactic capacity. RESULTS: We adapted an agarose spot migration assay as an in vitro system to evaluate the cell recruitment capacity of locally delivered or localized EVs. Cell migration was tracked for 12 h or 48 h, respectively. Thereafter, endpoint migration images and time-lapse videos were analysed to quantify several parameters aiming to determine the migration of cells to either MSC-EV or pro-metastatic EV. The number of cells contained inside the agarose spots, the migration distance, the area occupied by cells, the directionality of the cell movement, and the Euclidean distance were measured. This multi-parametric evaluation revealed the potential of different MSC-EV preparations to recruit endothelial cells and to detect an enhanced recruitment capacity of highly metastatic PC3-derived EVs (PC3-EVs) compared to low-metastatic LNCaP-EVs in a tumour cell-specific manner. CONCLUSIONS: Overall, this agarose spot migration assay may offer a diversity of measurements and migration settings not provided by classical migration assays and reveal its potential use in the EV field in two different contexts with recruitment in common: regeneration and cancer metastasis.

Soluble mutant huntingtin drives early human pathogenesis in Huntington's disease.

Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of new-born mice. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connexions within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed soluble forms of mHTT, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT monomers and oligomers, which were internalised by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT soluble forms with key cellular organelles initially drives disease progression in HD patients and their transmission through exosomes contributes to spread the disease in a non-cell autonomous manner.

Mesenchymal stromal cells induced regulatory B cells are enriched in extracellular matrix genes and IL-10 independent modulators.

Regulatory B cells (Breg) are essential players in tolerance and immune homeostasis. However, lack of specific Breg markers limit their potential in clinical settings. Mesenchymal stromal cells (MSC) modulate B cell responses and are described to induce Breg in vitro. The aim of this work was to characterize MSC induced Breg (iBreg) and identify specific Breg biomarkers by RNAseq. After 7-day coculture with adipose tissue-derived MSC, B cells were enriched in transitional B cell populations, with increased expression and secretion of IL-10 and no TNFα. In addition, iBreg showed potential to modulate T cell proliferation at 2 to 1 cell ratios and their phenotype remained stable for 72h. RNAseq analysis of sorted IL-10 positive and negative iBreg populations identified over 1500 differentially expressed genes (DEG) among both populations. Analysis of biological processes of DEG highlighted an enrichment of immune regulation and extracellular matrix genes in IL-10- iBreg populations, while IL-10+ iBreg DEG were mostly associated with cell activation. This was supported by T cells modulation assays performed in the presence of anti-IL-10 neutralizing antibodies showing the non-essential role of IL-10 in the immunomodulatory capacity of iBregs on T cells. However, based on RNAseq results we explored the role of TGF-ß and found out that it plays a major role on iBreg induction and iBreg immunomodulatory properties. Therefore, we report that MSC induce B cell populations characterized by the generation of extracellular matrix and immune modulation independently of IL-10.

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV-Cell Interaction and Functional Activation of Endothelial Cells.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions.

Acellular cardiac scaffolds enriched with MSC-derived extracellular vesicles limit ventricular remodelling and exert local and systemic immunomodulation in a myocardial infarction porcine model.

Rationale: Extracellular vesicles (EVs) from mesenchymal stromal cell (MSC) are a potential therapy for cardiac healing after myocardial infarction (MI). Nevertheless, neither their efficient administration nor therapeutic mechanisms are fully elucidated. Here, we evaluate the preclinical efficacy of a tissue engineering approach to locally deliver porcine cardiac adipose tissue MSC-EV (cATMSC-EV) in an acute MI pig model. Methods: After MI by permanent ligation of the coronary artery, pigs (n = 24) were randomized to Untreated or treated groups with a decellularised pericardial scaffold filled with peptide hydrogel and cATMSC-EV purified by size exclusion chromatography (EV-Treated group) or buffer (Control group), placed over the post-infarcted myocardium. Results: After 30 days, cardiac MRI showed an improved cardiac function in EV-Treated animals, with significantly higher right ventricle ejection fraction (+20.8% in EV-Treated; p = 0.026), and less ventricle dilatation, indicating less myocardial remodelling. Scar size was reduced, with less fibrosis in the distal myocardium (-42.6% Col I in EV-Treated vs Untreated; p = 0.03), a 2-fold increase in vascular density (EV-Treated; p = 0.019) and less CCL2 transcription in the infarct core. EV-treated animals had less macrophage infiltration in the infarct core (-31.7% of CD163+ cells/field in EV-Treated; p = 0.026), but 5.8 times more expressing anti-inflammatory CD73 (p = 0.015). Systemically, locally delivered cATMSC-EV also triggered a systemic effect, doubling the circulating IL-1ra (p = 0.01), and reducing the PBMC rush 2d post-MI, the TNFα and GM-CSF levels at 30d post-MI, and modulating the CD73+ and CCR2+ monocyte populations, related to immunomodulation and fibrosis modulation. Conclusions: These results highlight the potential of cATMSC-EV in modulating hallmarks of ischemic injury for cardiac repair after MI.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

In Vitro Characterization of Human CD24hiCD38hi Regulatory B Cells Shows CD9 Is Not a Stable Breg Cell Marker.

Regulatory B (Breg) cells are endowed with immune suppressive functions. Various human and murine Breg subtypes have been reported. While interleukin (IL)-10 intracellular staining remains the most reliable way to identify Breg cells, this technique hinders further essential functional studies. Recent findings suggest that CD9 is an effective surface marker of murine IL-10 competent Breg cells. However, the stability of CD9 and its relevance as a unique marker for human Breg cells, which have been widely characterized as CD24hiCD38hi, have not been investigated. Here, we demonstrate that CD9 expression is sensitive to in vitro B cell stimulations. CD9 expression could either be re-expressed or downregulated in purified CD9-negative B cells and CD9-positive B cells, respectively. We found no significant differences in the Breg differentiation capacity of the CD9-negative and CD9-positive B cells. Furthermore, CD9-positive B cells co-express CD40 and CD86, suggesting their nature as B cell activation or co-stimulatory molecules, rather than regulatory ones. Therefore, we report the relatively unstable CD9 as a distinct surface molecule, indicating the need for further research for a more reliable marker to purify human Breg cells.

B Cell-Derived Extracellular Vesicles Reveal Residual B Cell Activity in Kidney Graft Recipients Undergoing Pre-Transplant Desensitization.

Background: Living-donor kidney transplant (LDKT) recipients undergoing desensitization for Human Leukocyte Antigen (HLA)-incompatibility have a high risk of developing antibody-mediated rejection (ABMR). The purpose of the study is to evaluate if residual B cell activity after desensitization could be estimated by the presence of circulating B cell-derived extracellular vesicles (BEVs). Methods: BEVs were isolated by Sepharose-based size exclusion chromatography and defined as CD19+ and HLA-II+ extracellular vesicles. We analyzed stored serum samples from positive crossmatch LDKT recipients before and after desensitization at first post-transplant biopsy and at 12-month protocol biopsy (n = 11). Control groups were formed by hypersensitized patients who were not submitted to desensitization (n = 10) and by low-risk recipients (n = 9). A prospective validation cohort of 11 patients also included the analysis of B cells subpopulations in recipients' blood and lymph nodes recovered upon graft implantation, along with BEVs analysis before and after desensitization. Results: We found out that CD19+ and HLA-II+BEVs dropped significantly after desensitization and relapse in patients who later developed ABMR was evident. We validated these findings in a proof-of-concept prospective cohort of 6 patients who received the same desensitization protocol and also in a control group of 5 LDKT recipients. In these patients, B cell subpopulations were also studied in recipients' blood and lymph nodes that were recovered before the graft implantation. We confirmed the significant drop in BEVs after desensitization and that this paralleled the reduction in CD19+cells in lymph nodes, while in peripheral blood B cells, this change was almost undetectable. Conclusions: BEVs reflected B cell residual activity after desensitization and this could be a valid surrogate of humoral alloreactivity in this setting.

Potential of Extracellular Vesicle-Associated TSG-6 from Adipose Mesenchymal Stromal Cells in Traumatic Brain Injury.

Multipotent mesenchymal stromal cells (MSC) represent a promising strategy for a variety of medical applications. Although only a limited number of MSC engraft and survive after in vivo cellular infusion, MSC have shown beneficial effects on immunomodulation and tissue repair. This indicates that the contribution of MSC exists in paracrine signaling, rather than a cell-contact effect of MSC. In this review, we focus on current knowledge about tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and mechanisms based on extracellular vesicles (EV) that govern long-lasting immunosuppressive and regenerative activity of MSC. In this context, in particular, we discuss the very robust set of findings by Jha and colleagues, and the opportunity to potentially extend their research focus on EV isolated in concentrated conditioned media (CCM) from adipose tissue derived MSC (ASC). Particularly, the authors showed that ASC-CCM mitigated visual deficits after mild traumatic brain injury in mice. TSG-6 knockdown ASC were, then, used to generate TSG-6-depleted CCM that were not able to replicate the alleviation of abnormalities in injured animals. In light of the presented results, we envision that the infusion of much distilled ASC-CCM could enhance the alleviation of visual abnormalities. In terms of EV research, the advantages of using size-exclusion chromatography are also highlighted because of the enrichment of purer and well-defined EV preparations. Taken together, this could further delineate and boost the benefit of using MSC-based regenerative therapies in the context of forthcoming clinical research testing in diseases that disrupt immune system homeostasis.

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Extracellular Vesicles From Liver Progenitor Cells Downregulates Fibroblast Metabolic Activity and Increase the Expression of Immune-Response Related Molecules.

Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.

Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors.

BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. METHODS: uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. RESULTS: Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs.

Low doses of LPS exacerbate the inflammatory response and trigger death on TLR3-primed human monocytes.

TLR sensing of pathogens triggers monocyte activation to initiate the host innate immune response to infection. Monocytes can dynamically adapt to different TLR agonists inducing different patterns of inflammatory response, and the sequence of exposure to TLRs can dramatically modulate cell activation. Understanding the interactions between TLR signalling that lead to synergy, priming and tolerance to TLR agonists may help explain how prior infections and inflammatory conditioning can regulate the innate immune response to subsequent infections. Our goal was to investigate the role of MyD88-independent/dependent TLR priming on modulating the monocyte response to LPS exposure. We stimulated human blood monocytes with agonists for TLR4 (LPS), TLR3 (poly(I:C)) and TLR7/8 (R848) and subsequently challenged them to low doses of endotoxin. The different TLR agonists promoted distinct inflammatory signatures in monocytes. Upon subsequent LPS challenge, LPS- and R848-primed monocytes did not enhance the previous response, whereas poly(I:C)-primed monocytes exhibited a significant inflammatory response concomitant with a sharp reduction on cell viability. Our results show that TLR3-primed monocytes are prompted to cell death by apoptosis in the presence of low endotoxin levels, concurrent with the production of high levels of TNFα and IL6. Of note, blocking of TNFR I/II in those monocytes did reduce TNFα production but did not abrogate cell death. Instead, direct signalling through TLR4 was responsible of such effect. Collectively, our study provides new insights on the effects of cross-priming and synergism between TLR3 and TLR4, identifying the selective induction of apoptosis as a strategy for TLR-mediated host innate response.

Graphene oxide enhances alginate encapsulated cells viability and functionality while not affecting the foreign body response.

The combination of protein-coated graphene oxide (GO) and microencapsulation technology has moved a step forward in the challenge of improving long-term alginate encapsulated cell survival and sustainable therapeutic protein release, bringing closer its translation from bench to the clinic. Although this new approach in cell microencapsulation represents a great promise for long-term drug delivery, previous studies have been performed only with encapsulated murine C2C12 myoblasts genetically engineered to secrete murine erythropoietin (C2C12-EPO) within 160 µm diameter hybrid alginate protein-coated GO microcapsules implanted into syngeneic mice. Here, we show that encapsulated C2C12-EPO myoblasts survive longer and release more therapeutic protein by doubling the micron diameter of hybrid alginate-protein-coated GO microcapsules to 380 µm range. Encapsulated mesenchymal stem cells (MSC) genetically modified to secrete erythropoietin (D1-MSCs-EPO) within 380 µm-diameter hybrid alginate-protein-coated GO microcapsules confirmed this improvement in survival and sustained protein release in vitro. This improved behavior is reflected in the hematocrit increase of allogeneic mice implanted with both encapsulated cell types within 380 µm diameter hybrid alginate-protein-coated GO microcapsules, showing lower immune response with encapsulated MSCs. These results provide a new relevant step for the future clinical application of protein-coated GO on cell microencapsulation.

Mesenchymal Stem Cells Induce Expression of CD73 in Human Monocytes In Vitro and in a Swine Model of Myocardial Infarction In Vivo.

The ectoenzymes CD39 and CD73 regulate the purinergic signaling through the hydrolysis of adenosine triphosphate (ATP)/ADP to AMP and to adenosine (Ado), respectively. This shifts the pro-inflammatory milieu induced by extracellular ATP to the anti-inflammatory regulation by Ado. Mesenchymal stem cells (MSCs) have potent immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype aiding tissue repair. In vitro, we observed that human cardiac adipose tissue-derived MSCs (cATMSCs) and umbilical cord MSCs similarly polarize monocytes toward a regulatory M2 phenotype, which maintained the expression of CD39 and induced expression of CD73 in a cell contact dependent fashion, correlating with increased functional activity. In addition, the local treatment with porcine cATMSCs using an engineered bioactive graft promoted the in vivo CD73 expression on host monocytes in a swine model of myocardial infarction. Our results suggest the upregulation of ectonucleotidases on MSC-conditioned monocytes as an effective mechanism to amplify the long-lasting immunomodulatory and healing effects of MSCs delivery.

Inflammatory Conditions Dictate the Effect of Mesenchymal Stem or Stromal Cells on B Cell Function.

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43- selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC.

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low-density lipoprotein receptor-related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet-free plasma by enzyme-linked immunosorbent assay. Plasma-derived EVs were extracted by size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo-transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin-3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9- and CD81-positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin-3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro.

Giardia intestinalis (G.I), is an anaerobic protozoan and the aetiological agent of giardiasis, a diarrhoea present worldwide and associated with poverty. G.I has a simple life cycle alternating between cyst and trophozoite. Cysts are transmitted orally to the stomach and transform to trophozoites in the intestine by a multifactorial process. Recently, microvesicles (MVs) have been found to be released from a wide range of eukaryotic cells. We have observed a release of MVs during the life cycle of G.I., identifying MVs from active trophozoites and from trophozoites differentiating to the cyst form. The aim of the current work was to investigate the role of MVs from G.I in the pathogenesis of giardiasis. MVs from log phase were able to increase the attachment of G. intestinalis trophozoites to Caco-2 cells. Moreover, MVs from G. intestinalis could be captured by human immature dendritic cells, resulting in increased activation and allostimulation of human dendritic cells. Lipid rafts participate in the MV biogenesis and in the attachment to Caco-2 cells. Nevertheless, proteomic analysis from two types of MVs has shown slight differences at the protein levels. An understanding of biogenesis and content of MVs derived from trophozoites might have important implications in the pathogenesis of the disease.

Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine.

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.

Serum-derived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins.

PRRSV is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide and limitations in vaccinology. Exosomes are 30-100 nm vesicles of endocytic origin. Remarkably, immunizations with exosomes containing antigens from tumors or pathogens are capable of eliciting protective immune responses, albeit variably, in cancer and infectious diseases. Here we describe the isolation, molecular composition and immunogenicity of serum-derived exosomes from naïve animals, from PRRSV viremic animals and from animals previously PRRSV infected but already free of viruses (non viremic). Exosomes were isolated through size exclusion chromatography and characterized by different methodologies. Exosome-enriched fractions from naïve and natural infected animals contained classical tetraspanin exosomal markers (CD63 and CD81) and high concentrations of particles in the size-range of exosomes as detected by nanoparticle tracking analysis and cryo-TEM. NanoLC-MS/MS was used to identify viral antigens associated to exosomes. PRRSV-proteins were detected in serum samples from only viremic animals and from animals previously infected already free of viruses (non-viremic), but not in controls. Moreover, immune sera from pigs previously exposed to PRRSV specifically reacted against exosomes purified from non-viremic pig sera in a dose-dependent manner, a reactivity not detected when naïve sera was used in the assay. To facilitate future studies, a scaling-up process was implemented. To the best of our knowledge, this is the first molecular characterization of serum-derived exosomes from naïve pigs and pigs actively or previously infected with PRRSV. The presence of antigenic viral proteins in serum-derived exosomes free of virus, suggest their use as a novel vaccine approach against PRRSV.