Microvesicles from Mesenchymal Stem Cells Overexpressing MiR-34a Ameliorate Renal Fibrosis In Vivo.

INTRODUCTION: We recently discovered that microvesicles (MVs)  derived from mesenchymal stem cells (MSCs) overexpressing  miRNA-34a can alleviate experimental kidney injury in mice. In  this study, we further explored the effects of miR34a-MV on renal  fibrosis in the unilateral ureteral obstruction (UUO) models.  Methods. Bone marrow MSCs were modified by lentiviruses  overexpressing miR-34a, and MVs were collected from the  supernatants of MSCs. C57BL6/J mice were divided into control,  unilateral ureteral obstruction (UUO), UUO + MV, UUO + miR-34aMV and UUO + miR-34a-inhibitor-MV groups. MVs were injected  to mice after surgery. The mice were then euthanized on day 7  and 14 of modeling, and renal tissues were collected for further  analyses by Hematoxylin and eosin, Masson's trichrome,  and Immunohistochemical (IHC) staining.  Results. The UUO + MV group exhibited a significantly reduced  degree of renal interstitial fibrosis with inflammatory cell infiltration,  tubular epithelial cell atrophy, and vacuole degeneration compared  with the UUO group. Surprisingly, overexpressing miR-34a enhanced  these effects of MSC-MV on the UUO mice.  Conclusion. Our study demonstrates that miR34a further enhances  the effects of MSC-MV on renal fibrosis in mice through the  regulation of epithelial-to-mesenchymal transition (EMT) and  Notch pathway. miR-34a may be a candidate molecular therapeutic  target for the treatment of renal fibrosis. DOI: 10.52547/ijkd.7673.

Nano-seq analysis reveals different functional tendency between exosomes and microvesicles derived from hUMSC.

BACKGROUND: Extracellular vesicles (EVs) from human umbilical cord mesenchymal stem cells (hUMSCs) are widely considered to be the best mediators for cell-free therapy. An understanding of their composition, especially RNA, is particularly important for the safe and precise application of EVs. Up to date, the knowledge of their RNA components is limited to NGS sequencing and cannot provide a comprehensive transcriptomic landscape, especially the long and full-length transcripts. Our study first focused on the transcriptomic profile of hUMSC-EVs based on nanopore sequencing. METHODS: In this study, different EV subtypes (exosomes and microvesicles) derived from hUMSCs were isolated and identified by density gradient centrifugation. Subsequently, the realistic long transcriptomic profile in different subtypes of hUMSC-EVs was systematically compared by nanopore sequencing and bioinformatic analysis. RESULTS: Abundant transcript variants were identified in EVs by nanopore sequencing, 69.34% of which transcripts were fragmented. A series of full-length and long transcripts was also observed and showed a significantly higher proportion of intact or near-complete transcripts in exosomes than that in microvesicles derived from hUMSCs. Although the composition of RNA biotypes transported by different EV subtypes was similar, the distribution of transcripts and genes revealed the inter-heterogeneity and intra-stability between exosomes and microvesicles. Further, 85 different expressed transcripts (56 genes) and 7 fusion genes were identified. Pathway enrichment analysis showed that upregulated-expressed genes in microvesicles were mainly enriched in multiple neurodegenerative diseases, while upregulated-expressed genes in exosomes were mainly enriched in neutrophil extracellular trap formation, suggesting different functional tendencies of EV subtypes. CONCLUSIONS: This study provides a novel understanding of different types of hUMSC-EVs, which not only suggests different transcriptome sorting mechanisms between exosomes and microvesicles, but also shows that different EV subtypes from the same source have different physiological functions, suggesting distinct clinical application prospects.

Cellular and Molecular Nature of Fragmentation of Human Embryos.

Embryo fragmentation represents a phenomenon generally characterized by the presence of membrane-bound extracellular cytoplasm into the perivitelline space. Recent evidence supports the cellular and molecular heterogeneity of embryo fragments. In this narrative review, we described the different embryo fragment-like cellular structures in their morphology, molecular content, and supposed function and have reported the proposed theories on their origin over the years. We identified articles related to characterization of embryo fragmentation with a specific literature search string. The occurrence of embryo fragmentation has been related to various mechanisms, of which the most studied are apoptotic cell death, membrane compartmentalization of altered DNA, cytoskeletal disorders, and vesicle formation. These phenomena are thought to result in the extrusion of entire blastomeres, release of apoptotic bodies and other vesicles, and micronuclei formation. Different patterns of fragmentation may have different etiologies and effects on embryo competence. Removal of fragments from the embryo before embryo transfer with the aim to improve implantation potential should be reconsidered on the basis of the present observations.

Extracellular vesicles derived from hypoxia-preconditioned olfactory mucosa mesenchymal stem cells enhance angiogenesis via miR-612.

Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3'-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer.

Estradiol-driven metabolism in transwomen associates with reduced circulating extracellular vesicle microRNA-224/452.

OBJECTIVE: Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice. METHODS: Following plasma miR-sequencing (seq) in a transwomen discovery (n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes. RESULTS: Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose). CONCLUSION: This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy.

Methods for the Detection of Circulating Pseudogenes and Their Use as Cancer Biomarkers.

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles.

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Adipocyte-secreted microvesicle-derived miR-148a regulates adipogenic and osteogenic differentiation by targeting Wnt5a/Ror2 pathway.

AIMS: Adipocyte-secreted microvesicles (MVs)-derived microRNAs (miRNAs) are relevant to adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteonecrosis of the femoral head (ONFH). Our aims are to investigate the mechanism of adipocyte-derived MVs-miR-148a in ONFH. MATERIALS AND METHODS: Adipocyte-derived MVs were identified via transmission electron microscopy and specific markers expression. The adipogenic and osteogenic differentiation were investigated by Oil-Red O staining, alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining and osteogenic or adipogenic factors levels. Genes and proteins expression were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The relationship between miR-148a and Wnt5a was tested via dual-luciferase reporter analysis. The adipogenic differentiation and osteogenic differentiation in methylprednisolone (MPS)-induced ONFH rat model were assessed via hematoxylin-eosin (HE) staining, and immunohistochemical staining of collagen I (COL I). KEY FINDINGS: Adipocyte-derived MVs promoted adipogenic differentiation via increasing Oil-Red O staining positive cells, adiponectin (Adipoq), acid-binding protein 2 (aP2) and peroxisome proliferator-activated receptor γ (PPAR-γ) levels, and repressed osteogenic differentiation of BMSCs via decreasing ARS staining positive cells, ALP, Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) levels. MiR-148a was present in adipocyte-derived MVs, and miR-148a knockdown inhibited adipogenic differentiation and promoted osteogenic differentiation. Furthermore, Wnt5a expression was regulated by miR-148a. MiR-148a overexpression facilitated adipogenic differentiation and suppressed osteogenic differentiation via regulating the Wnt5a/Ror2 pathway. Adipocyte-derived MVs promoted adipogenic differentiation and inhibited osteogenic differentiation in MPS-induced ONFH rat model. SIGNIFICANCE: Adipocyte-derived MVs-miR-148a promoted adipogenic differentiation and suppressed osteogenic differentiation via targeting the Wnt5a/Ror2 pathway.

Keratinocyte-derived microvesicle particles mediate ultraviolet B radiation-induced systemic immunosuppression.

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.

Centrifugation Removes a Population of Large Vesicles, or "Macroparticles," Intermediate in Size to RBCs and Microvesicles.

Microparticles or microvesicles (MPs/MVs) are sub-cellular vesicles with a growing number of known biological functions. Microvesicles from a variety of parent cells within the vascular system increase in numerous pathological states. Red blood cell-derived MVs (RMVs) are relatively less studied than other types of circulating MVs despite red blood cells (RBCs) being the most abundant intravascular cell. This may be in part due the echoes of past misconceptions that RBCs were merely floating anucleate bags of hemoglobin rather than dynamic and responsive cells. The initial aim of this study was to maximize the concentration of RMVs derived from various blood or blood products by focusing on the optimal isolation conditions without creating more MVs from artificial manipulation. We found that allowing RBCs to sediment overnight resulted in a continuum in size of RBC membrane-containing fragments or vesicles extending beyond the 1 µm size limit suggested by many as the maximal size of an MV. Additionally, dilution and centrifugation factors were studied that altered the resultant MV population concentration. The heterogeneous size of RMVs was confirmed in mice models of hemolytic anemia. This methodological finding establishes a new paradigm in that it blurs the line between RBC, fragment, and RMV as well as suggests that the concentration of circulating RMVs may be widely underestimated given that centrifugation removes the majority of such RBC-derived membrane-containing particles.

Circulating extracellular vesicles release oncogenic miR-424 in experimental models and patients with aggressive prostate cancer.

Extracellular vesicles (EVs) are relevant means for transferring signals across cells and facilitate propagation of oncogenic stimuli promoting disease evolution and metastatic spread in cancer patients. Here, we investigated the release of miR-424 in circulating small EVs or exosomes from prostate cancer patients and assessed the functional implications in multiple experimental models. We found higher frequency of circulating miR-424 positive EVs in patients with metastatic prostate cancer compared to patients with primary tumors and BPH. Release of miR-424 in small EVs was enhanced in cell lines (LNCaPabl), transgenic mice (Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG) and patient-derived xenograft (PDX) models of aggressive disease. EVs containing miR-424 promoted stem-like traits and tumor-initiating properties in normal prostate epithelial cells while enhanced tumorigenesis in transformed prostate epithelial cells. Intravenous administration of miR-424 positive EVs to mice, mimicking blood circulation, promoted miR-424 transfer and tumor growth in xenograft models. Circulating miR-424 positive EVs from patients with aggressive primary and metastatic tumors induced stem-like features when supplemented to prostate epithelial cells. This study establishes that EVs-mediated transfer of miR-424 across heterogeneous cell populations is an important mechanism of tumor self-sustenance, disease recurrence and progression. These findings might indicate novel approaches for the management and therapy of prostate cancer.

Platelet microparticles load a repertory of miRNAs programmed to drive osteogenic phenotype.

Autologous platelet-rich plasma accelerates bone healing by releasing biomolecules during their degranulation process, which are transported by vesicle-like structures called platelet microparticles (PMPs). However, the underlying mechanisms regulating the osteogenic differentiation by PMP-released miRs remain poorly understood and this prompted us to better address this issue. Thus, miRNAseq expression profiles (E-GEOD-76789) were downloaded from ArrayExpress database. GEO2R was performed to evaluate the differential expression, and mirnatap R package was used to find targets for differentially expressed miRNAs. An extend protein-protein (ePPI) network for osteogenic marker proteins was generated using String, and DAVID tools were used to perform gene ontology and KEGG pathway analysis from ePPI and miRNAs targets. Our data show that ePPI network was composed by 232 nodes and 2,175 edges, with a clustering coefficient of 0.546. MCODE was able to identify seven clusters contained in the ePPI network, and the two that presented a score above 10 were used in further analysis. Conversely, 15,944 different targets were found as down-expressed while 5,715 different targets were up-expressed. Among the downregulated 75 miRNAs, 70 have predicted targets present in the ePPI network, while the 21 upregulated miRNAs have 19 predicted targets in the ePPI network. Our study provides a registry of miRNAs that play a central role in regulating osteogenic phenotype, which might have potential therapeutic applications in bone regeneration and bone tissue engineering.

STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain.

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon ß expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase-dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.

Extracellular microvesicles/exosomes: discovery, disbelief, acceptance, and the future?

There are concepts in science that need time to overcome initial disbelief before finally arriving at the moment when they are embraced by the research community. One of these concepts is the biological meaning of the small, spheroidal vesicles released from cells, which are described in the literature as microparticles, microvesicles, or exosomes. In the beginning, this research was difficult, as it was hard to distinguish these small vesicles from cell debris or apoptotic bodies. However, they may represent the first language of cell-cell communication, which existed before a more specific intercellular cross-talk between ligands and receptors emerged during evolution. In this review article, we will use the term "extracellular microvesicles" (ExMVs) to refer to these small spheroidal blebs of different sizes surrounded by a lipid layer of membrane. We have accepted an invitation from the Editor-in-Chief to write this review in observance of the 20th anniversary of the 2001 ASH Meeting when our team demonstrated that, by horizontal transfer of several bioactive molecules, including mRNA species and proteins, ExMVs harvested from embryonic stem cells could modify hematopoietic stem/progenitor cells and expand them ex vivo. Interestingly, the result that moved ExMV research forward was published first in 2005 in Leukemia, having been previously rejected by other major scientific journals out of simple disbelief. Therefore, the best judge of a new concept is the passage of time, although the speed of its adoption is aided by perseverance and confidence in one's own data. In this perspective article, we will provide a brief update on the current status of, hopes for, and likely future of ExMV research as well as therapeutic and diagnostic applications, with a special emphasis on hematopoiesis.

Novel Mechanism of Microvesicle Regulation by the Antiviral Protein Tetherin During HIV Infection.

Background Microvesicles are cell membrane-derived vesicles that have been shown to augment inflammation. Specifically, monocyte-derived microvesicles (MDMVs), which can express the coagulation protein tissue factor, contribute to thrombus formation and cardiovascular disease. People living with HIV experience higher prevalence of cardiovascular disease and also exhibit increased levels of plasma microvesicles. The process of microvesicle release has striking similarity to budding of enveloped viruses. The surface protein tetherin inhibits viral budding by physically tethering budding virus particles to cells. Hence, we investigated the role of tetherin in regulating the release of MDMVs during HIV infection. Methods and Results The plasma of aviremic HIV-infected individuals had increased levels of tissue factor + MDMVs, as measured by flow cytometry, and correlated to reduced tetherin expression on monocytes. Superresolution confocal and electron microscopy showed that tetherin localized at the site of budding MDMVs. Mechanistic studies revealed that the exposure of monocytes to HIV-encoded Tat triggered tetherin loss and subsequent rise in MDMV production. Overexpression of tetherin in monocytes led to morphologic changes in the pseudopodia directly underneath the MDMVs. Further, tetherin knockout mice demonstrated a higher number of circulating MDMVs and less time to bleeding cessation. Conclusions Our studies define a novel regulatory mechanism of MDMV release through tetherin and explore its contribution to the procoagulatory state that is frequently observed in people with HIV. Such insights could lead to improved therapies for individuals infected with HIV and also for those with cardiovascular disease.

Ectosomal PKM2 Promotes HCC by Inducing Macrophage Differentiation and Remodeling the Tumor Microenvironment.

Tumor-derived extracellular vesicles are important mediators of cell-to-cell communication during tumorigenesis. Here, we demonstrated that hepatocellular carcinoma (HCC)-derived ectosomes remodel the tumor microenvironment to facilitate HCC progression in an ectosomal PKM2-dependent manner. HCC-derived ectosomal PKM2 induced not only metabolic reprogramming in monocytes but also STAT3 phosphorylation in the nucleus to upregulate differentiation-associated transcription factors, leading to monocyte-to-macrophage differentiation and tumor microenvironment remodeling. In HCC cells, sumoylation of PKM2 induced its plasma membrane targeting and subsequent ectosomal excretion via interactions with ARRDC1. The PKM2-ARRDC1 association in HCC was reinforced by macrophage-secreted cytokines/chemokines in a CCL1-CCR8 axis-dependent manner, further facilitating PKM2 excretion from HCC cells to form a feedforward regulatory loop for tumorigenesis. In the clinic, ectosomal PKM2 was clearly detected in the plasma of HCC patients. This study highlights a mechanism by which ectosomal PKM2 remodels the tumor microenvironment and reveals ectosomal PKM2 as a potential diagnostic marker for HCC.

Clinical applications of extracellular vesicle long RNAs.

Extracellular vesicles (EVs), primarily exosomes and microvesicles, are critical intercellular mediators of communication. Over the past decade, improved knowledge and methodologies have enabled the detection and quantification of RNA species in EVs, despite their extremely low levels. Recently, EV-associated long RNAs (exLRs) have been drawing much attention. Delivered by EVs, they have higher stability, greater difference in temporal and spatial expression, and the capacity to remodel both proximal and distal recipient cells. These properties guarantee their roles as biomarkers, therapeutic targets, vaccines, and gene therapy agents in a wide range of human diseases. Despite the progress in this area of research, limitations in both knowledge and methodologies have hindered its further development. Herein, we comprehensively reviewed studies related to exLRs, including protein-coding messenger RNAs (mRNAs) and noncoding RNAs (long noncoding RNAs and circular RNAs) in EVs to indicate their value in the diagnosis and treatment of human diseases; we also present a series of yet unsettled issues in this novel area, hence providing insights for future studies.

Platelet factor 4 and ß-thromboglobulin mRNAs in circulating microparticles of trauma patients as diagnostic markers for deep vein thrombosis.

Deep vein thrombosis (DVT) is a common complication after trauma. The development of markers to predict DVT in trauma patients is needed, and circulating microparticles (MPs) and their contents are possible candidates. In this study, we aimed to identify platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) mRNAs in circulating MPs as potential markers for DVT diagnosis in trauma patients. Fifteen trauma patients diagnosed with DVT and fifteen matched patients without DVT were included in this study. Fifteen healthy volunteers also were included as controls. Circulating MPs were obtained from the plasma of all study subjects. Annexin V+ MPs and platelet-derived MPs (PMPs) were quantified using flow cytometry. PF4 and ß-TG mRNAs in MPs were determined by qPCR, and the common logarithm of relative quantitation (RQ) was calculated using the comparative Ct method. Receiver-operating characteristic (ROC) curves were performed to analyze the diagnostic value of PF4 and ß-TG mRNAs. No significant differences were found in Annexin V+ MPs and PMPs levels between trauma patients with and without DVT. However, both PF4 and ß-TG mRNAs in MPs from the DVT group were significantly higher than the non-DVT group and healthy controls (P = 0.014 for PF4, P = 0.010 for ß-TG). The ROC curve analysis showed that both the PF4 mRNA (area-under curve (AUC) 0.756, P = 0.017) and the ß-TG mRNA (AUC 0.751, P = 0.019) had a positive predictive value for DVT. This finding indicates that the PF4 and ß-TG mRNAs in MPs may be used as potential biomarkers for DVT diagnosis in trauma patients.

Human extracellular vesicles and correlation with two clinical forms of toxoplasmosis.

AIM: This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS: Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS: Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION: These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis.

Microparticles from tumors exposed to radiation promote immune evasion in part by PD-L1.

Radiotherapy induces immune-related responses in cancer patients by various mechanisms. Here, we investigate the immunomodulatory role of tumor-derived microparticles (TMPs)-extracellular vesicles shed from tumor cells-following radiotherapy. We demonstrate that breast carcinoma cells exposed to radiation shed TMPs containing elevated levels of immune-modulating proteins, one of which is programmed death-ligand 1 (PD-L1). These TMPs inhibit cytotoxic T lymphocyte (CTL) activity both in vitro and in vivo, and thus promote tumor growth. Evidently, adoptive transfer of CTLs pre-cultured with TMPs from irradiated breast carcinoma cells increases tumor growth rates in mice recipients in comparison with control mice receiving CTLs pre-cultured with TMPs from untreated tumor cells. In addition, blocking the PD-1-PD-L1 axis, either genetically or pharmacologically, partially alleviates TMP-mediated inhibition of CTL activity, suggesting that the immunomodulatory effects of TMPs in response to radiotherapy is mediated, in part, by PD-L1. Overall, our findings provide mechanistic insights into the tumor immune surveillance state in response to radiotherapy and suggest a therapeutic synergy between radiotherapy and immune checkpoint inhibitors.