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Objectives Detecting occult cancer in patients with unprovoked venous thromboembolism (VTE) remains a significant challenge. Our objective was to investigate the potential predictive role of coagulation-related biomarkers in the diagnosis of occult malignancies. Methods We conducted a nested case-control study with a 1-year prospective cohort of 214 patients with unprovoked VTE, with a focus on identifying occult cancer. At the time of VTE diagnosis, we measured various biomarkers, including soluble P-selectin (sP-selectin), dimerized plasmin fragment D (D-dimer), platelets, leukocytes, hemoglobin, total extracellular vesicles (EVs), EVs expressing tissue factor on their surface (TF+EVs), and EVs expressing P-selectin on their surface (Psel+EVs) in all participants. Results We observed statistically significant increased levels of sP-selectin (P =.015) in patients with occult cancer. Despite an increase in Psel+EVs, TF+EVs, D-dimer, and platelets within this group, however, no significant differences were found. When sP-selectin exceeded 62 ng/mL and D-dimer surpassed 10,000 µg/L, the diagnosis of occult cancer demonstrated a specificity of up to 91% (95% CI, 79.9%-96.7%). Conclusions The combination of sP-selectin and D-dimer can be a valuable biomarker in detecting occult cancer in patients with unprovoked VTE. Further research is necessary to ascertain whether easily measurable biomarkers such as sP-selectin and D-dimer can effectively distinguish between patients who have VTE with and without hidden malignancies. [ABSTRACT FROM AUTHOR]

Particles fabricated from red blood cells (RBCs) can serve as vehicles for delivery of various biomedical cargos. Flipping of phosphatidylserine (PS) from the inner to the outer membrane leaflet normally occurs during the fabrication of such particles. PS externalization is a signal for phagocytic removal of the particles from circulation. Herein, we demonstrate that membrane cholesterol enrichment can mitigate the outward display of PS on microparticles engineered from RBCs. Our in-vitro results show that the phagocytic uptake of cholesterol-enriched particles by murine macrophages takes place at a lowered rate, resulting in reduced uptake as compared to RBC-derived particles without cholesterol enrichment. When administered via tail-vein injection into healthy mice, the percent of injected dose (ID) per gram of extracted blood for cholesterol-enriched particles was ∼1.5 and 1.8 times higher than the particles without cholesterol enrichment at 4 and 24 h, respectively. At 24 h, ∼43% ID/g of the particles without cholesterol enrichment was eliminated or metabolized while ∼94% ID/g of the cholesterol-enriched particles were still retained in the body. These results indicate that membrane cholesterol enrichment is an effective method to reduce PS externalization on the surface of RBC-derived particles and increase their longevity in circulation.

Recently, we described a phenomenon whereby apoptotic cells generate and release CrkI-containing microvesicles, which stimulate proliferation in surrounding cells upon contact to compensate for their own demise. We termed these microvesicles "ACPSVs" for Apoptotic Compensatory Proliferation Signaling microvesicles. As immune cells and a majority of current cancer therapeutics destroy tumor cells primarily by apoptosis, we conducted a small pilot study to assess the possibility that ACPSVs may also be generated in squamous cell carcinomas. We first evaluated a primary and a metastatic squamous cell carcinoma cancer cell lines for their ability to produce ACPSVs under normal and apoptotic conditions. We next conducted a pilot study to assess the occurrence of ACPSVs in solid tumors extracted from 20 cancer patients with squamous cell carcinomas. Both cancer cell lines produced copious amounts of ACPSVs under apoptotic conditions. Interestingly, the metastatic squamous cell carcinoma cancer cell line also produced high levels of ACPSVs under healthy condition, suggesting that the ability to generate ACPSVs may be hijacked by these cells. Importantly, ACPSVs were also abundant in the solid tumors of all squamous cell carcinoma cancer patients. Detection of ACPSVs in cancer has potentially important ramifications in tumor biology and cancer therapeutics which warrants further investigation.

• Patients who were candidates for heart valve surgery with lower presurgical levels of platelet-derived microparticles (PMPs) received more RBC transfusion within the first 24 hours. • The presurgical number of PMPs, hemoglobin level, and red blood cell (RBC) count were identified as risk factors for the need for RBC transfusion. • Preoperative assessment of MPs levels may be helpful in transfusion management. Cell-derived microparticles (MPs) are membrane vesicles that have emerged as a potential biomarker for various diseases and their clinical complications. This study investigates the role of MPs as a risk factor for blood transfusion in patients with valve heart disease undergoing cardiac surgery. Forty adult patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled, and venous blood samples were collected prior to surgical incision. Plasma rich in MPs was prepared by double centrifugation, and the concentration of MPs was determined using the Bradford method. Flow cytometry analysis was performed to determine MPs count and phenotype. Patients were divided into "with transfusion" (n = 18) and "without transfusion" (n = 22) groups based on red blood cell (RBC) transfusion. There was no significant difference in MPs concentration between the "with transfusion" and "without transfusion" groups. Although the count of preoperative platelet-derived MPs (PMPs), monocyte-derived MPs (MMPs), and red cell-derived MPs (RMPs) was higher in "without transfusion" group, these differences were not statistically significant. The preoperative PMPs count was negatively correlated with RBC transfusion (P = 0.005, r = -0.65). Multivariate logistic regression analysis revealed that the count of CD41+ PMPs, Hemoglobin (Hb), and RBC count were risk factors for RBC transfusion. This study suggests that the presurgical levels of PMPs, Hb, and RBC count can serve as risk factors of RBC transfusion in patients with valve heart disease undergoing cardiac surgery. The findings provide insights into the potential use of MPs as biomarkers for blood transfusion prediction in cardiac surgery. [ABSTRACT FROM AUTHOR]

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.

The tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca2+ mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.

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.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 μM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.

Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol-rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane-derived particles are enriched in 'accessible cholesterol' (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.

Despite laparoscopy being a standardized option to diagnose pelvic endometriotic implants, non-invasive biomarkers are necessary to avoid the discomfort of invasive procedures. Recent evidence suggests a potential role of microRNAs (miRNAs) as feasible biomarkers for the early diagnosis of endometriosis. Following the recommendations in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, we systematically searched PubMed, EMBASE, Scopus, Cochrane Library, and Science Direct in January 2023. We provided no restriction on the country and year of publication, and considered English published articles. We selected studies including patients with endometriosis and describing miRNA regulation in the context of endometriosis. Overall, 45 studies fulfilled the inclusion criteria, and 2045 patients with endometriosis and 1587 controls were screened. Patients were analyzed concerning miRNAs expression and sources, stage of disease, and symptoms, and compared to controls. Among DEMs, the ones with the widest delta between endometriosis patients and controls—Relative Expression ≥ 4 Log2(ratio)—were miR-145, miR-191, miR-195, miR-21-5p, miR-106b-5p, miR-195-5p, miR-451a, miR-200c, miR-20a-5p, and miR-15a-5p. Although the epigenetic regulation is partially unclear, miRNAs are valid biomarkers to diagnose endometriotic lesions in symptomatic and non-symptomatic women. MiRNAs modulation should be clarified, especially during therapies or relapse, to plan targeted management protocols. [ABSTRACT FROM AUTHOR]

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.

Nucleic acid binding polymers (NABPs) have been extensively used as vehicles for DNA and RNA delivery. More recently, we discovered that a subset of these NABPs can also serve as anti-inflammatory agents by capturing pro-inflammatory extracellular nucleic acids and associated protein complexes that promote activation of toll-like receptors (TLRs) in diseases such as lupus erythematosus. Nucleic-acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here, we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic-acid-mediated activation of TLRs and invasion of PC tumor cells in vitro, but can also directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

Protein delivery is often used in tissue engineering applications to control differentiation processes, but is limited by protein instability and cost. An alternative approach is to control the cellular microenvironment through biomaterial-mediated sequestration of cell-secreted proteins important to differentiation. Thus, we utilized heparin-based microparticles to modulate cellular differentiation via protein sequestration in an in vitro model system of endochondral ossification. Heparin and poly(ethylene-glycol) (PEG; a low-binding material control)-based microparticles were incorporated into ATDC5 cell spheroids or incubated with ATDC5 cells in transwell culture. Reduced differentiation was observed in the heparin microparticle group as compared to PEG and no microparticle-containing groups. To determine if observed changes were due to sequestration of cell-secreted protein, the proteins sequestered by heparin microparticles were analyzed using SDS-PAGE and mass spectrometry. It was found that heparin microparticles bound insulin-like growth factor binding proteins (IGFBP)-3 and 5. When incubated with a small-molecule inhibitor of IGFBPs, NBI 31772, a similar delay in differentiation of ATDC5 cells was observed. These results indicate that heparin microparticles modulated chondrocytic differentiation in this system via sequestration of cell-secreted protein, a technique that could be beneficial in the future as a means to control cellular differentiation processes. STATEMENT OF SIGNIFICANCE:In this work, we present a proof-of-principle set of experiments in which heparin-based microparticles are shown to modulate cellular differentiation through binding of cell-secreted protein. Unlike existing systems that rely on expensive protein with limited half-lives to elicit changes in cellular behavior, this technique focuses on temporal modulation of cell-generated proteins. This technique also provides a biomaterials-based method that can be used to further identify sequestered proteins of interest. Thus, this work indicates that glycosaminoglycan-based biomaterial approaches could be used as substitutes or additions to traditional methods for modulating and identifying the cell-secreted proteins involved in directing cellular behavior.

Tumor-cell-derived microparticles (TMPs) have been recognized as chemotherapeutic drug carriers and immunomodulators for anti-tumor therapy. Research in the clinical application of TMPs has been devoted to developing an effective delivery formulation that could enhance their therapeutic effects. Here, we propose thermal-responsive agarose hydrogel microspheres (MTX-TMPs@MSs) with encapsulation of Methotrexate (MTX)-packaging TMPs (MTX-TMPs) and black phosphorus quantum dots (BPQDs) by microfluidic technology for synergistic treatment of malignant ascites. The laden MTX-TMPs, separated from apoptotic tumor cells, could target tumor cells for the delivery of chemotherapy drugs and modulate the tumor immune microenvironment. Under near-infrared (NIR) induced thermal stimulation, MTX-TMPs could be controllably released from the low-melting-point agarose matrix hydrogel microspheres for chemotherapy (CHT) and immunotherapy (IMT). In addition, benefiting from photothermal therapy (PTT)-induced tumor immunogenic death, the anti-tumor immune response triggered by MTX-TMPs was further enhanced. Based on these features, the MTX-TMPs@MSs could remarkably eliminate tumor cells in vitro and obviously suppress tumor growth in vivo through synergistic PTT, CHT, and IMT. Therefore, it is envisaged that this TMPs-integrated microcarrier will have promising applications in clinical tumor therapy. STATEMENT OF SIGNIFICANCE: Primary liver cancer ranks third among the causes of cancer deaths globally, with hepatocellular carcinoma (HCC) being the most common type. In particular, patients with advanced HCC accompanied by malignant ascites, a common complication, indicate tumor metastasis and a poor prognosis. In this paper, we developed stimuli-responsive hydrogel microspheres from microfluidics for the delivery of methotrexate (MTX)-loaded tumor-cell-derived microparticles (MTX-TMPs) for synergistic chemotherapy, photothermal therapy, and immunotherapy. The release of MTX-TMPs from hydrogel microspheres could be on-demand controlled through BPQDs-mediated photothermal stimulus. On the other hand, BPQDs-mediated mild hyperthermia cooperatesss with MTX-TMPs-induced chemotherapy could participate in remodeling the tumor immunosuppressive microenvironment. Thus, the prepared microcarrier system holds great promise for tumor therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Inc. All rights reserved.)

Besides enhancing osseo- and soft tissue integration, modulating inflammation at the implant site is also crucial for dental implant success. Uncontrolled peri-implant inflammation can cause significant loss of surrounding tissue and implant failure. It was recently shown that microvesicles (MVs), a less-studied type of extracellular vesicles, play a crucial role in cell-to-cell communication and may modulate angiogenesis and inflammatory response. The effect of MVs on regulating inflammation at an implant site, however, remains unexplored. In the current study, MVs were isolated and characterised from human primary gingival fibroblasts (hGFs) and loaded within titania nanotubes (TNTs, fabricated via anodisation on 3D Ti wire implants) towards their local release. The modified implants were characterised using SEM and confocal imaging to confirm the loading and local release of MVs from TNTs. In vitro studies demonstrated the internalisation of hGFs-MVs by human gingival keratinocytes (OKF6/TERT2 cell line), which caused a significant reduction in the production of pro-inflammatory cytokines. The results support MVs-releasing TNTs as a promising implant surface modification strategy to reduce inflammation, paving the way for further advancements in therapeutic dental implants., (© 2023. The Author(s).)

AIM: To evaluate differences in microparticle profiles in vitreous samples between diabetic and non-diabetic eyes undergoing vitrectomy. METHODS: Un-masked cross-sectional series of 34 eyes undergoing vitrectomy. Vitreous specimens were collected and processed to evaluate for membrane integrity (DAPI), apoptosis (Annexin-V), and endothelial-cell origin (V-Cadherin). A BD LSR II flow cytometer was used for analysis and standardized sub-micron-sized beads were used for size comparison. RESULTS: Thirty-four specimens underwent analysis. Greater levels of Annexin-V were found on microparticles from specimens in which blood had entered the vitreous (n=12) compared to those without blood (n=22; 52.3%±30.7% vs 19.6%±27.2%, P=0.002). Patients with diabetes having surgery with hemorrhage (n=7) had greater expression of Annexin-V than those without hemorrhage (n=8; 62.1%±31.7% vs 18.9%±20.9%, P=0.009). However, in patients with non-diabetic vitreous hemorrhage, the level of Annexin-V expression was not significantly different compared to other disease processes (38.6%±25.7%, n=5 vs 20.0%±30.9%, n=14, P=0.087). CONCLUSION: Increased expression of the apoptotic marker, Annexin-V is detected on vitreous microparticles in diabetes-related vitreous hemorrhage. When evaluating vitreous hemorrhage in patients without diabetes, the apoptotic signal is not significantly different. Vitrectomy in patients with diabetes, and improvement in visual outcomes, may be related to the removal of a serum-derived, pro-apoptotic vitreous. Further investigation is warranted in order to identify the molecular characteristics of microparticles that regulate disease.

Extracellular vesicles (EVs) released by virtually every cell of all organisms are involved in processes of intercellular communication through the delivery of their functional mRNAs, proteins and bioactive lipids. We previously demonstrated that mouse embryonic stem cell-released EVs (mESEVs) are able to transfer their content to different target retinal cells, inducing morphological and biochemical changes in them. The main objective of this paper is to characterize EVs derived from human embryonic stem cells (hESEVs) and investigate the effects that they have on cultured retinal glial, progenitor Müller cells, which are known to give rise to retinal neurons under specific conditions. This would allow us to establish if hESEVs have a pro-regenerative potential not yet described that could be used in the future for treatment of human retinal degenerative diseases. Initially, we showed that hESEVs are heterogeneous in size, contain mRNAs and proteins involved in the induction and maintenance of stem cell pluripotency and can be internalized by cultured Müller cells. After a single exposure to hESEVs these cells display changes in their gene expression profile, and with multiple exposures they de-differentiate and trans-differentiate into retinal neuronal precursors. hESEVs were then fractionated into microvesicles (MVs) and exosomes (EXOs), which were characterized by size, specific surface proteins and biochemical/molecular components. We demonstrate that despite the similar internalization of non-fractionated hESEVs, MVs and EXOs by Müller progenitor cells, in vitro, only the release of MVs' cargo into the cells' cytoplasm induces specific changes in their levels of pluripotency mRNAs and early retinal proteins. EXOs do not produce any detectable effect. Thus, we conclude that MVs and MVs-containing hESEVs are promising agents that possibly could promote the regeneration of diseased or damaged retinas in vivo through inducing glial Müller cells to become replacement neurons.

In the CNS, microglia are activated in response to injury or infection and in neurodegenerative diseases. The endocytic and cell signaling receptor, LDL receptor-related protein-1 (LRP1), is reported to suppress innate immunity in macrophages and oppose microglial activation. The goal of this study was to identify novel mechanisms by which LRP1 may regulate microglial activation. Using primary cultures of microglia isolated from mouse brains, we demonstrated that LRP1 gene silencing increases expression of proinflammatory mediators; however, the observed response was modest. By contrast, the LRP1 ligand, receptor-associated protein (RAP), robustly activated microglia, and its activity was attenuated in LRP1-deficient cells. An important element of the mechanism by which RAP activated microglia was its ability to cause LRP1 shedding from the plasma membrane. This process eliminated cellular LRP1, which is anti-inflammatory, and generated a soluble product, shed LRP1 (sLRP1), which is potently proinflammatory. Purified sLRP1 induced expression of multiple proinflammatory cytokines and the mRNA encoding inducible nitric-oxide synthase in both LRP1-expressing and -deficient microglia. LPS also stimulated LRP1 shedding, as did the heat-shock protein and LRP1 ligand, calreticulin. Other LRP1 ligands, including α2-macroglobulin and tissue-type plasminogen activator, failed to cause LRP1 shedding. Treatment of microglia with a metalloproteinase inhibitor inhibited LRP1 shedding and significantly attenuated RAP-induced cytokine expression. RAP and sLRP1 both caused neuroinflammation in vivo when administered by stereotaxic injection into mouse spinal cords. Collectively, these results suggest that LRP1 shedding from microglia may amplify and sustain neuroinflammation in response to proinflammatory stimuli.

ObjectiveCoagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood.Approach and resultsHere, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation.ConclusionsThese data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.

The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.

BackgroundIt is well established that as a blood unit ages, fewer of the unit's red blood cells (RBCs) remain in circulation post-transfusion. The mechanism for clearance is not well defined. Phosphatidylethanolamine (PE) is a phospholipid that is primarily found on the inner leaflet of healthy cells, and is an important ligand for phagocytosis of dead cells when exposed.ObjectivesThe objective of the present study was to measure the change in PE exposure in donor RBCs over increasing storage ages using the novel PE-specific probe, duramycin.MethodsFive adsol (AS-1) preserved RBC units were sampled weekly for 6 weeks and were labelled with duramycin. The percentage of PE exposed on red cells in each sample was determined using flow cytometry. Surface phosphatidylserine (PS) was evaluated for comparison.ResultsWe found that RBCs in AS-preserved donor units increasingly exposed PE, from less than 1% in freshly processed RBCs, to nearly 20% at 42 days of storage and correlated with increased relative vesiculation or microparticle concentration and release of cell-free haemoglobin. By comparison, only 5% of cells exposed PS at 42 days.ConclusionWe conclude that exposure of PE in the RBC outer membrane was higher than that of PS during 42 days of storage and correlated significantly with increased vesiculation and release of haemoglobin.

Background: Cell-derived microparticles (MPs) as membrane vesicles are procoagulant. They play a role in surgical hemostasis. In this study, the correlations between the circulating level of cell-derived MPs and surgical variables in heart valve surgery were investigated. Methods: The present prospective case-series study was conducted in Rajaie Cardiovascular Medical and Research Center from January through March 2021. Forty patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled. Before the induction of anesthesia and 30 minutes after the administration of protamine sulfate, venous blood samples were collected. After MP isolation, the concentration of MPs was determined via the Bradford method. Flow cytometry analysis was performed to determine the MP count and phenotype. Intraoperative variables and postoperative routine coagulation tests were defined as surgical variables. Postoperative coagulopathy was defined as an activated partial thromboplastin time (aPTT) ≥48 seconds or an international normalized ratio (INR) >1.5. Results: The total concentration of MPs and the MP count increased significantly after surgery compared with before surgery. The postoperative concentration of MPs was positively correlated with the CPB time (P=0.030, ρ=0.40). The preoperative concentration of MPs was significantly lower in patients with higher postoperative aPTT and INR (P=0.003, P= -0.50 and P=0.020, P= -0.40, respectively). In multivariate logistic regression analysis, the preoperative MP concentration (OR, 1.00; 95% CI, 1.00 to 1.01; P=0.017) was considered a risk factor for postoperative coagulopathy. Conclusion: The levels of MPs, especially platelet-derived MPs, rose after surgery, in correlation with the CPB time. Given the role of MPs in the induction of coagulation and inflammation, they can be considered therapeutic goals for preventing postoperative complications. In addition, the preoperative levels of MPs are a risk factor for predicting the occurrence of postoperative coagulopathy in heart valve surgery. [ABSTRACT FROM AUTHOR]

Background: Cell-derived microparticles (MPs) as membrane vesicles are procoagulant. They play a role in surgical hemostasis. In this study, the correlations between the circulating level of cell-derived MPs and surgical variables in heart valve surgery were investigated. Methods: The present prospective case-series study was conducted in Rajaie Cardiovascular Medical and Research Center from January through March 2021. Forty patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled. Before the induction of anesthesia and 30 minutes after the administration of protamine sulfate, venous blood samples were collected. After MP isolation, the concentration of MPs was determined via the Bradford method. Flow cytometry analysis was performed to determine the MP count and phenotype. Intraoperative variables and postoperative routine coagulation tests were defined as surgical variables. Postoperative coagulopathy was defined as an activated partial thromboplastin time (aPTT) ≥48 seconds or an international normalized ratio (INR) >1.5. Results: The total concentration of MPs and the MP count increased significantly after surgery compared with before surgery. The postoperative concentration of MPs was positively correlated with the CPB time (P=0.030, ρ=0.40). The preoperative concentration of MPs was significantly lower in patients with higher postoperative aPTT and INR (P=0.003, Ρ= −0.50 and P=0.020, Ρ= −0.40, respectively). In multivariate logistic regression analysis, the preoperative MP concentration (OR, 1.00; 95% CI, 1.00 to 1.01; P=0.017) was considered a risk factor for postoperative coagulopathy. Conclusion: The levels of MPs, especially platelet-derived MPs, rose after surgery, in correlation with the CPB time. Given the role of MPs in the induction of coagulation and inflammation, they can be considered therapeutic goals for preventing postoperative complications. In addition, the preoperative levels of MPs are a risk factor for predicting the occurrence of postoperative coagulopathy in heart valve surgery.

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However, the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular, interferon-γ (IFN-γ)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-γ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation, the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore, the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-γ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-γ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture, whereas IDO expression by IFN-γ-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore, sustained IDO expression induced by IFN-γ-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-γ-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether, microparticle delivery of IFN-γ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237.

RationaleObese children are at increased risk for developing obstructive sleep apnea (OSA), and both of these conditions are associated with an increased risk for endothelial dysfunction (ED) in children, an early risk factor for atherosclerosis and cardiovascular disease. Although weight loss and treatment of OSA by adenotonsillectomy improve endothelial function, not every obese child or child with OSA develops ED. Exosomes are circulating extracellular vesicles containing functional mRNA and microRNA (miRNA) that can be delivered to other cells, such as endothelial cells.ObjectivesTo investigate whether circulating exosomal miRNAs of children with OSA differentiate based on endothelial functional status.MethodsObese children (body mass index z score >1.65) and nonobese children were recruited and underwent polysomnographic testing (PSG), and fasting endothelial function measurements and blood draws in the morning after PSG. Plasma exosomes were isolated from all subjects. Isolated exosomes were then incubated with confluent endothelial cell monolayer cultures. Electric cell-substrate impedance sensing systems were used to determine the ability of exosomes to disrupt the intercellular barrier formed by confluent endothelial cells. In addition, immunofluorescent assessments of zonula occludens-1 tight junction protein cellular distribution were conducted to examine endothelial barrier dysfunction. miRNA and mRNA arrays were also applied to exosomes and endothelial cells, and miRNA inhibitors and mimics were transfected for mechanistic assays.Measurements and main resultsPlasma exosomes isolated from either obese children or nonobese children with OSA were primarily derived from endothelial cell sources and recapitulated ED, or its absence, in naive human endothelial cells and also in vivo when injected into mice. Microarrays identified a restricted signature of exosomal miRNAs that readily distinguished ED from normal endothelial function. Among the miRNAs, expression of exosomal miRNA-630 was reduced in children with ED and normalized after therapy along with restoration of endothelial function. Conversely, transfection of exosomes from subjects without ED with an miRNA-630 inhibitor induces the ED functional phenotype. Gene target discovery experiments further revealed that miRNA-630 regulates 416 gene targets in endothelial cells that include the Nrf2, AMP kinase, and tight junction pathways.ConclusionsThese observations elucidate a novel role of exosomal miRNA-360 as a putative key mediator of vascular function and cardiovascular disease risk in children with underlying OSA and/or obesity, and identify therapeutic targets.

Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells.

Cancer survivors face a variety of challenges as they cope with disease recurrence and a myriad of normal tissue complications brought on by radio- and chemotherapeutic treatment regimens. For patients subjected to cranial irradiation for the control of CNS malignancy, progressive and debilitating cognitive dysfunction remains a pressing unmet medical need. Although this problem has been recognized for decades, few if any satisfactory long-term solutions exist to resolve this serious unintended side effect of radiotherapy. Past work from our laboratory has demonstrated the neurocognitive benefits of human neural stem cell (hNSC) grafting in the irradiated brain, where intrahippocampal transplantation of hNSC ameliorated radiation-induced cognitive deficits. Using a similar strategy, we now provide, to our knowledge, the first evidence that cranial grafting of microvesicles secreted from hNSC affords similar neuroprotective phenotypes after head-only irradiation. Cortical- and hippocampal-based deficits found 1 mo after irradiation were completely resolved in animals cranially grafted with microvesicles. Microvesicle treatment was found to attenuate neuroinflammation and preserve host neuronal morphology in distinct regions of the brain. These data suggest that the neuroprotective properties of microvesicles act through a trophic support mechanism that reduces inflammation and preserves the structural integrity of the irradiated microenvironment.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.

Optoelectronic tweezers (OET) has advanced within the past decade to become a promising tool for cell and microparticle manipulation. Its incompatibility with high conductivity media and limited throughput remain two major technical challenges. Here a novel manipulation concept and corresponding platform called Self-Locking Optoelectronic Tweezers (SLOT) are proposed and demonstrated to tackle these challenges concurrently. The SLOT platform comprises a periodic array of optically tunable phototransistor traps above which randomly dispersed single cells and microparticles are self-aligned to and retained without light illumination. Light beam illumination on a phototransistor turns off the trap and releases the trapped cell, which is then transported downstream via a background flow. The cell trapping and releasing functions in SLOT are decoupled, which is a unique feature that enables SLOT's stepper-mode function to overcome the small field-of-view issue that all prior OET technologies encountered in manipulation with single-cell resolution across a large area. Massively parallel trapping of more than 100,000 microparticles has been demonstrated in high conductivity media. Even larger scale trapping and manipulation can be achieved by linearly scaling up the number of phototransistors and device area. Cells after manipulation on the SLOT platform maintain high cell viability and normal multi-day divisibility.

Extracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation [UC], precipitation [EQ‐O, EQ‐TC], size exclusion chromatography [SEC], and ultrafiltration [UF]) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non‐vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool‐derived EVs as a source for novel biomarkers for early CRC detection. [ABSTRACT FROM AUTHOR]

Background: Microparticles (MPs) are small vesicles released from the cell membrane. Accordingly, these contain active molecules to mediate biological processes, including cell proliferation and cell cycle progression. The fusion of myeloma cell lines with immunized B lymphocytes is a critical step of hybridoma technology. MPs modulate lymphoproliferation, thereby facilitating B cell expansion and successful immortalization. Human alloimmune B cells are considered valuable sources of monoclonal antibodies, and peripheral blood is a pool of B lymphocytes. The present study aimed to investigate the role of myeloma-derived MPs in the proliferation of alloimmune peripheral blood mononuclear cells (PBMCs). Materilas and Methods: In the current experimental study, ultracentrifugation isolated MPs from three different myeloma cell lines, including U266, P3x63Ag8, and SP2/0. PBMCs were extracted from the whole blood of a patient with thalassemia alloimmune exposed to MPs. Thereafter, both the proliferation and cell viability were evaluated using inverted microscopy and the trypan blue staining method. Results: MPs derived from the P3X63Ag8 myeloma cell line were shown to have the most effects on cell proliferation and viability (p=0.0005). MPs of the SP2/0 cell line initially increased the proliferation of PBMCs, but viable cells were drastically reduced in the following weeks. As well, U266 cell-derived MPs increased the proliferation of PBMCs (p=0.0001), but it was half of the group receiving P3x63Ag8 derived MPs. However, the viability of treated cells remained almost constant for 5-weeks. Conclusion: Altogether, the obtained data indicated that P3X63Ag8 and U266 derived MPs could increase the viability of PBMCs. If the complimentary examination is confirmed, these MPs could also be used as the potential agents in B lymphocyte proliferation, thereby helping in immortalization and antibody production. [ABSTRACT FROM AUTHOR]

Hong-Peng Chen,1,2,* Xiao-Yan Wang,1,* Xiao-Yan Pan,1 Wang-Wang Hu,1 Shu-Ting Cai,1 Kiran Joshi,3 Lie-Hua Deng,1,2 Daqing Ma3 1Department of Critical Care Medicine, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, People’s Republic of China; 2Zhanjiang Key Laboratory of Organ Injury and Protection and Translational Medicine, Zhanjiang, Guangdong, People’s Republic of China; 3Division of Anesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK*These authors contributed equally to this workCorrespondence: Lie-Hua Deng; Daqing MaDepartment of Critical Care Medicine, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, ChinaEmail glinson@126.com; d.ma@imperial.ac.ukIntroduction: Because of its high morbidity and mortality, sepsis remains the leading cause of death in the ICU. Microparticles (MP) have been largely studied as potential diagnostic or prognostic markers in various diseases including sepsis.Objective: The biological and clinical relevance of neutrophil-derived microparticles (NDMPs) within the MP population remains unclear. The objective of this study was to elucidate the relationship between plasma NDMPs and the prognosis of patients with sepsis and/or septic shock.Methods: The study was designed as an observational, noninterventional clinical study. The cohort for this study included 40 sepsis and 40 septic shock patients together with 10 healthy controls admitted to the Intensive Care Unit (ICU) and the Health Surveillance Center in the Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, China, from January to November 2018, respectively. The degree of critical disease for sepsis and septic shock was evaluated, with data analyses conducted from 2018 to 2019.Results: On days 1, 3 and 5 post-admission a series of data including plasma NDMP levels, patient demographics, TNF-α levels, IL-6 levels, sTREM-1 levels, and the sepsis severity score measurements were collected. A survival curve was plotted against levels of plasma NDMPs. Levels of NDMPs were observed to be higher in the septic shock patients than in the sepsis patients on days 1, 3, and 5 post-ICU admission (p < 0.05). NDMP levels were significantly increased in sepsis and septic shock patients with a parallel increase in pro-inflammatory mediators and sepsis severity score (p < 0.05) as well as mortality.Conclusion: Our data suggest that NDMPs may be a biomarker of sepsis severity and mortality although its implications on sepsis prognosis warrant further study.Keywords: sepsis, septic shock, cell-derived microparticles, biomarker, prognosis

Abstract Background Lower‐leg injury and knee arthroscopy are both associated with venous thromboembolism (VTE). The mechanism of VTE in both situations is unknown, including the role of procoagulant microparticles. This may provide useful information for individualizing thromboprophylactic treatment in both patient groups. Objective We aimed to study the effect of (1) lower‐leg trauma and (2) knee arthroscopy on procoagulant phospholipid‐dependent (PPL) activity plasma levels. Methods POT‐(K)CAST trial participants who did not develop VTE were randomly selected for the current study. Plasma was collected shortly after lower‐leg trauma or before and after knee arthroscopy. For aim 1, samples of 67 patients with lower‐leg injury were compared with control samples (preoperative samples of 74 patients undergoing arthroscopy). Linear regression was used to obtain mean ratios (natural logarithm retransformed data), adjusted for age, sex, body mass index, infections, and comorbidities. For aim 2, pre‐ and postoperative samples of 49 patients undergoing arthroscopy were compared using paired t tests. PPL activity was measured using modified activated factor X–dependent PPL clotting assay. Results For aim 1, PPL activity levels were almost threefold higher in patients with lower‐leg injury compared with controls, that is, mean ratio, 2.82 (95% confidence interval [CI], 1.98‐4.03). For aim 2, postoperative PPL activity levels did not change significantly, that is, mean change, −0.72 mU/mL (95% CI, −2.03 to 0.59). Conclusion Lower‐leg trauma was associated with increased plasma levels of PPL activity, in contrast to knee arthroscopy. Lower‐leg trauma triggers the release of procoagulant microparticles.

Extracellular Vesicles (EVs) have received considerable attention in recent years, both as mediators of intercellular communication pathways that lead to tumor progression, and as potential sources for discovery of novel cancer biomarkers. For many years, research on EVs has mainly investigated either the mechanism of biogenesis and cargo selection and incorporation, or the methods of EV isolation from available body fluids for biomarker discovery. Recent studies have highlighted the existence of different populations of cancer-derived EVs, with distinct molecular cargo, thus pointing to the possibility that the various EV populations might play diverse roles in cancer and that this does not happen randomly. However, data attributing cancer specific intercellular functions to given populations of EVs are still limited. A deeper functional, biochemical and molecular characterization of the various EV classes might identify more selective clinical markers, and significantly advance our knowledge of the pathogenesis and disease progression of many cancer types.

Pre-eclampsia is one of the main causes of maternal morbidity in the world. At present, it may be related to insufficient recasting of uterine spiral arterioles, excessive activation of inflammation and immunity, damage of endothelial vascular cells, and so on. Recent studies have shown that microparticle can increase the expression of NF-κB in patients with pre -eclampsia, and promote the formation of neutrophil extracellular trap, resulting in inflammation at the maternal -fetal interface and vascular endothelial cell dysfunction, and participate in the pathophysiological process of pre-eclampsia. [ABSTRACT FROM AUTHOR]

The need to increase the donor pool for lung transplantation is a major public health issue. We previously found that administration of mesenchymal stem cells "rehabilitated" marginal donor lungs rejected for transplantation using ex vivo lung perfusion. However, the use of stem cells has some inherent limitation such as the potential for tumor formation. In the current study, we hypothesized that microvesicles, small anuclear membrane fragments constitutively released from mesenchymal stem cells, may be a good alternative to using stem cells. Using our well established ex vivo lung perfusion model, microvesicles derived from human mesenchymal stem cells increased alveolar fluid clearance (i.e. ability to absorb pulmonary edema fluid) in a dose-dependent manner, decreased lung weight gain following perfusion and ventilation, and improved airway and hemodynamic parameters compared to perfusion alone. Microvesicles derived from normal human lung fibroblasts as a control had no effect. Co-administration of microvesicles with anti-CD44 antibody attenuated these effects, suggesting a key role of the CD44 receptor in the internalization of the microvesicles into the injured host cell and its effect. In summary, microvesicles derived from human mesenchymal stem cells were as effective as the parent mesenchymal stem cells in rehabilitating marginal donor human lungs.

RationaleMicrovesicles (MVs) are anuclear fragments of cells released from the endosomal compartment or shed from surface membranes. We and other investigators demonstrated that MVs released by mesenchymal stem cells (MSCs) were as effective as the cells themselves in inflammatory injuries, such as after endotoxin-induced acute lung injury. However, the therapeutic effects of MVs in an infectious model of acute lung injury remain unknown.ObjectivesWe investigated the effects of human MSC MVs on lung inflammation, protein permeability, bacterial clearance, and survival after severe bacterial pneumonia.MethodsWe tested the effects of MVs derived from human MSCs on Escherichia coli pneumonia in mice. We also studied the interactions between MVs and human monocytes and human alveolar epithelial type 2 cells.Measurements and main resultsAdministration of MVs derived from human MSCs improved survival in part through keratinocyte growth factor secretion and decreased the influx of inflammatory cells, cytokines, protein, and bacteria in mice injured with bacterial pneumonia. In primary cultures of human monocytes or alveolar type 2 cells, the uptake of MVs was mediated by CD44 receptors, which were essential for the therapeutic effects. MVs enhanced monocyte phagocytosis of bacteria while decreasing inflammatory cytokine secretion and increased intracellular ATP levels in injured alveolar epithelial type 2 cells. Prestimulation of MSCs with a toll-like receptor 3 agonist further enhanced the therapeutic effects of the released MVs.ConclusionsMVs derived from human MSCs were as effective as the parent stem cells in severe bacterial pneumonia.

Coronary artery disease (CAD) is characterised by progressive atherosclerotic plaque leading to flow-limiting stenosis, while myocardial infarction (MI) occurs due to plaque rupture or erosion with abrupt coronary artery occlusion. Multiple inflammatory pathways influence plaque stability, but direct assessment of endothelial inflammation at the site of coronary artery stenosis has largely been limited to pathology samples or animal models of atherosclerosis. We describe a technique for isolating and characterising endothelial cells (ECs) and EC microparticles (EMPs) derived directly from the site of coronary artery plaque during balloon angioplasty and percutaneous coronary intervention. Coronary artery endothelial cells (CAECs) were identified using imaging flow cytometry (IFC), and individual CAEC and EMP expression of the pro-atherogenic adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) was assessed immediately following angioplasty. Patients with MI registered 73 % higher VCAM-1 expression on their CAECs and 79 % higher expression on EMPs compared to patients with stable CAD. In contrast, VCAM-1 expression was absent on ECs in the peripheral circulation from these same subjects. VCAM-1 density was significantly higher on CAECs and EMPs among patients with MI and positively correlated with markers of myocardial infarct size. We conclude that increased VCAM-1 expression on EC and formation of EMP at the site of coronary plaque is positively correlated with the extent of vascular inflammation in patients with myocardial infarction.

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway-dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.

Antiphospholipid syndrome is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies, along with occurrence of vascular thrombosis and pregnancy morbidity. The variety of antiphospholipid antibodies and their related mechanisms, as well as the behavior of disease in wide groups of patients, have led some authors to propose a differentiation of this syndrome into two independent entities: vascular and obstetric antiphospholipid syndrome. Thus, previous studies have discussed whether specific autoantibodies may be responsible for this differentiation or, in contrast, how the same antibodies are able to generate two different clinical presentations. This discussion is yet to be settled. The capability of serum IgG from patients with vascular thrombosis to trigger the biogenesis of endothelial cell-derived microparticles in vitro is one of the previously discussed differences between the clinical entities of antiphospholipid syndrome. These vesicles constitute a prothrombotic mechanism as they can directly lead to clot activation in murine models and recalcified human plasma. Nevertheless, other indirect mechanisms by which microparticles can spread a procoagulant phenotype could be critical to understanding their role in antiphospholipid syndrome. For this reason, questions regarding the cargo of microparticles, and the signaling pathways involved in their biogenesis, are of interest in attempting to explain the behavior of this autoimmune disease. [ABSTRACT FROM AUTHOR]

In the last decade, the number of studies and publications on extracellular vesicles (EV) and exosomes has boomed. Colombia has displayed interest and progress in their study as shown in the increase of research project publications and products. However, this research field is still developing and has its own analytical challenges and technical limitations. For planning research projects and developing EV studies it is necessary to consider what is the state of the scientific field worldwide concerning EV nomenclature and classification, available techniques, resources, requirements and quality specifications, and the institutions that regulate the field. Answering this question will elicit EV studies that comply with international standards and respond to institutional demands and recommendations. However, the scientific information available is scattered and not all the aspects are considered in full. In this update, the available information is condensed and the official terms and currently defined nomenclature is presented, as well as the evolution of the field, the homogenization of the experimental parameters, the establishment of scientific authorities, institutions, and resources, and the recommendations generated worldwide for their development and research including their isolation, characterization, and functional studies. Finally, I analyzed the national context in a critical way, considering institutional strengths, common mistakes, and available analytical techniques and technologies. [ABSTRACT FROM AUTHOR]

Microvesicles are of increasing interest in biology as part of normal function of numerous systems; from the immune system (T cell activation) to implantation of the embryo (invasion of the trophoblasts) and sperm maturation (protein transfer in the epididymis). Yet, the mechanisms involved in the appearance of apical blebbing from healthy cells as part of their normal function remain understudied. Microvesicles are produced via one of two pathways: exocytosis or apical blebbing also termed ectocytosis. This work quantifies the histological appearance of apical blebbing in the porcine epididymis during development and examines the role of endogenous estrogens in regulating this blebbing. Apical blebbing appears at puberty and increases in a linear manner into sexual maturity suggesting that this blebbing is a mature phenotype. Endogenous estrogen levels were reduced with an aromatase inhibitor but such a reduction did not affect apical blebbing in treated animals compared with their vehicle-treated littermates. Epididymal production of apical blebs is a secretion mechanism of functionally mature principal cells regulated by factors other than estradiol.