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Aug 23, 2016

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Search terms: exosomes OR "extracellular vesicles" OR microvesicles OR microparticles. Direct link to the PubMed search here.

Prothrombotic mechanisms in patients with congenital p.Cys89Tyr mutation in CD59.

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Prothrombotic mechanisms in patients with congenital p.Cys89Tyr mutation in CD59.

Thromb Res. 2018 Jun 11;168:67-77

Authors: Tabib A, Hindi I, Karbian N, Zelig O, Falach B, Mevorach D

Abstract
BACKGROUND: Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome.
METHODS: Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9 μm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight.
FINDINGS: 2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2-2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients.
INTERPRETATION: Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

PMID: 29929138 [PubMed - as supplied by publisher]

Design and characterization of emulsified spray dried alginate microparticles as a carrier for the dually acting drug roflumilast.

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Design and characterization of emulsified spray dried alginate microparticles as a carrier for the dually acting drug roflumilast.

Eur J Pharm Sci. 2018 Jun 18;:

Authors: Mahmoud AA, Elkasabgy NA, Abdelkhalek AFA

Abstract
Roflumilast is a selective inhibitor of phosphodiesterase-4 isoenzyme in lung cells. Having psychiatric adverse reactions when administered orally affects negatively the patients' adherence to the drug. This work aimed to prepare emulsified spray dried alginate microparticles for the pulmonary delivery of roflumilast. Sodium alginate was used as microparticle-forming material, isopropyl myristate as an oil, Tween®80 as surfactant and calcium beta-glycerophosphate as cross-linking agent to enhance the mechanical properties of the particles. The prepared particles were evaluated for their encapsulation efficiency, particle size and in-vitro release. From the studied carriers, beta-cyclodextrin (CD) was the best regarding giving formulation smaller particle size and more sustained drug release. The inhalation profile of CD-based microparticles was investigated using Anderson cascade impactor. The aerosolization profile of CD-based microparticles suggested their efficiency to deliver the drug deep in the lung. The CD-based microparticles possessed more inhibitory effects on the viability of A549 cells and on the pro-inflammatory cytokines (TNF-α, IL-6 and IL-10) compared to the pure drug. Hence, CD-based microparticles could regulate the tumorigenesis besides tumor-associated inflammation. Finally, CD-based microparticles showed more sustained bronchodilatation properties in healthy human volunteers when compared to Ventolin®HFA. CD-based microparticles proved to be a promising carrier for inhaled roflumilast in human.

PMID: 29928985 [PubMed - as supplied by publisher]

Liquid biopsy in pancreatic cancer: the beginning of a new era.

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Liquid biopsy in pancreatic cancer: the beginning of a new era.

Oncotarget. 2018 Jun 01;9(42):26900-26933

Authors: Yadav DK, Bai X, Yadav RK, Singh A, Li G, Ma T, Chen W, Liang T

Abstract
With dismal survival rate pancreatic cancer remains one of the most aggressive and devastating malignancy. Predominantly, due to the absence of a dependable methodology for early identification and limited therapeutic options for advanced disease. However, it takes over 17 years to develop pancreatic cancer from initiation of mutation to metastatic cancer; therefore, if diagnosed early; it may increase overall survival dramatically, thus, providing a window of opportunity for early detection. Recently, genomic expression analysis defined 4 subtypes of pancreatic cancer based on mutated genes. Hence, we need simple and standard, minimally invasive test that can monitor those altered genes or their associated pathways in time for the success of precision medicine, and liquid biopsy seems to be one answer to all these questions. Again, liquid biopsy has an ability to pair with genomic tests. Additionally, liquid biopsy based development of circulating tumor cells derived xenografts, 3D organoids system, real-time monitoring of genetic mutations by circulating tumor DNA and exosome as the targeted drug delivery vehicle holds lots of potential for the treatment and cure of pancreatic cancer. At present, diagnosis of pancreatic cancer is frantically done on the premise of CA19-9 and radiological features only, which doesn't give a picture of genetic mutations and epigenetic alteration involved. In this manner, the current diagnostic paradigm for pancreatic cancer diagnosis experiences low diagnostic accuracy. This review article discusses the current state of liquid biopsy in pancreatic cancer as diagnostic and therapeutic tools and future perspectives of research in the light of circulating tumor cells, circulating tumor DNA and exosomes.

PMID: 29928492 [PubMed]

Impaired efferocytosis by monocytes in multiple myeloma.

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Impaired efferocytosis by monocytes in multiple myeloma.

Oncol Lett. 2018 Jul;16(1):409-416

Authors: Liang YY, Schwarzinger I, Simonitsch-Klupp I, Agis H, Oehler R

Abstract
Efficient clearance of apoptotic cells by efferocytosis is important for tissue homeostasis. Impaired efferocytosis leads to the accumulation of cell debris, which is regarded as a trigger in chronic inflammation and autoimmune diseases. Patients with hematological neoplastic disorders such as multiple myeloma (MM) exhibit high blood levels of apoptotic microparticles. The present study investigated whether these high levels of apoptotic microparticles are associated with insufficient dead cell clearance. Blood samples were collected from patients with MM immediately prior to and 3, 7 and 10 days after the initial cycle of bortezomib-based therapy. In addition, bone marrow aspirates (BMA) were collected prior to and following therapy. Prior to therapy, a 52% reduction in efferocytosis by blood monocytes was observed compared with the healthy controls (P<0.017). This was associated with an elevated number of 7-AAD+ dead cell remnants in the blood flow as well as in BMA. A portion of the blood samples contained active caspase 3. The subsequent bortezomib-based therapy had no effect on efferocytosis, although the quantity of dead cell remnants decreased. This reduction was associated with a decline in cluster of differentiation 8 (CD8)+ and CD4+ T cells and an increase in the number of monocytes. However, of 28 distinct soluble immune-modulating molecules (i.e. chemokines, cytokines and soluble co-stimulators) only C-C motif chemokine ligand 2 (CCL2), CCL24 and sCD27 were affected by bortezomib-based therapy. The levels of all other molecules remained unchanged or were below the detection threshold in all samples. The present study results revealed that the presence of dead cell remnants in the blood and bone morrow of patients with MM is associated with impaired efferocytosis by monocytes; however, its contribution to inflammatory events during MM remains unclear.

PMID: 29928429 [PubMed]

Exosome-mediated gefitinib resistance in lung cancer HCC827 cells via delivery of miR-21.

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Exosome-mediated gefitinib resistance in lung cancer HCC827 cells via delivery of miR-21.

Oncol Lett. 2018 Jun;15(6):9811-9817

Authors: Jing C, Cao H, Qin X, Yu S, Wu J, Wang Z, Ma R, Feng J

Abstract
Acquired resistance to gefitinib remains a major challenge in cancer treatment. In the present study, the effect of exosomes on the transmission of gefitinib resistance from gefitinib-resistant HCC827 lung cancer cells (H827R) to their gefitinib-sensitive counterparts and the potential underlying mechanisms by which this occurs was investigated. Exosomes were obtained from the cell supernatant using ultracentrifugation and the ExoQuick-TC exosome precipitation solution. Drug resistance was assessed by flow cytometry, apoptosis assays and cell counting kit-8 assays. The expression of microRNA (miR)-21 was analyzed by reverse transcription-quantitative polymerase chain reaction. Exosomes released by H827R cells (R/exo) may decrease the sensitivity of the human NSCLC HCC827 cell line to gefitinib. The results indicated that miR-21 expression was increased in R/exo and R/exo-treated H827S cells. However, miR-21 inhibition abrogated exosome-mediated drug resistance. Phosphorylated-protein kinase B (p-Akt), which is downstream of miR-21, was downregulated following gefitinib treatment; however, R/exo pretreatment elevated p-Akt levels and promoted the activation of Akt. By contrast, miR-21 inhibition reduced p-Akt expression. Therefore, the induction of miR-21 via exosomes and the activation of Akt may be mechanisms by which exosomes mediate the transfer of drug resistance.

PMID: 29928355 [PubMed]

Characteristics of Extracellular Vesicles in Red Blood Concentrates Change with Storage Time and Blood Manufacturing Method.

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Characteristics of Extracellular Vesicles in Red Blood Concentrates Change with Storage Time and Blood Manufacturing Method.

Transfus Med Hemother. 2018 May;45(3):185-193

Authors: Almizraq RJ, Holovati JL, Acker JP

Abstract
Background: Extracellular vesicles (EVs) in blood products are potential effectors of inflammation and coagulation after transfusion. The aim of this study was to assess the impact of different blood manufacturing methods and duration of hypothermic storage on the EV subpopulations in relation to other in vitro quality parameters of red blood cell concentrate (RCC) products.
Methods: RCCs were produced using whole blood filtration (WBF) or red cell filtration (RCF) (n = 12/method), refrigerated for 43 days, and evaluated for EV size profile and concentration, red cell deformability, ATP and 2,3-DPG, hemolysis, and hematological indices.
Results: The total number of EVs increased significantly with storage in both methods, and WBF-RCCs contained the higher numbers of EVs compared to RCF-RCCs. The concentration of small EVs was greater in WBF-RCCs versus RCF-RCCs, with difference between the two methods observed on day 43 of storage (p = 0.001). Throughout storage, significant decreases were identified in ATP, 2,3-DPG, and EImax, while an increase in hemolysis was observed in both RCC products.
Conclusion: The dynamic shift in the size and concentration of the EV subpopulations is dependent on the blood manufacturing method and length of storage. Better understanding of the potential clinical implications of these heterogeneous populations of EVs are needed.

PMID: 29928174 [PubMed]

Probing cellular mechanics with Acoustic Force Spectroscopy.

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Probing cellular mechanics with Acoustic Force Spectroscopy.

Mol Biol Cell. 2018 Jun 21;:mbcE18030154

Authors: Sorkin R, Bergamaschi G, Kamsma D, Brand G, Dekel E, Ofir-Birin Y, Rudik A, Gironella M, Ritort F, Regev-Rudzki N, Roos W, Wuite GJL

Abstract
A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, Red Blood Cell (RBC) deformability is key to their function, and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single cell methods provide very valuable information, they are often technically challenging and lack high data throughput needed to distinguish differences in heterogeneous populations, while fluid flow high throughput methods miss the accuracy to detect subtle differences. Here, we present a new method for multiplexed single-cell mechanical probing using Acoustic Force Spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular-vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision, and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

PMID: 29927358 [PubMed - as supplied by publisher]

A Method for Isolating and Characterizing Mesenchymal Stromal Cell-derived Extracellular Vesicles.

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A Method for Isolating and Characterizing Mesenchymal Stromal Cell-derived Extracellular Vesicles.

Curr Protoc Stem Cell Biol. 2018 Jun 11;:e55

Authors: Lo Sicco C, Reverberi D, Pascucci L, Tasso R

Abstract
The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages. The topics comprise description of the MSC-conditioned medium containing vesicles preparation, EV isolation, and characterization mainly by specifically set up flow cytometry and electron microscopy approaches, and in vitro methodologies involved in testing the EV anti-inflammatory capacity. The procedures described here can be easily reproduced and can be employed regardless of the type of progenitor cells used to secrete EVs. © 2018 by John Wiley & Sons, Inc.

PMID: 29927086 [PubMed - as supplied by publisher]

A Simply Modified Lymphocyte for Systematic Cancer Therapy.

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A Simply Modified Lymphocyte for Systematic Cancer Therapy.

Adv Mater. 2018 Jun 21;:e1801622

Authors: Zheng DW, Fan JX, Liu XH, Dong X, Pan P, Xu L, Zhang XZ

Abstract
Cytotherapy has received considerable attention in the field of cancer therapy, and various chemical or genetic methods have been applied to remold natural cells for improved therapeutic outcome of cytotherapy. A simple method to modify lymphocytes for cancer treatment by using a clinically used molecule, δ-aminolevulinic acid (δ-ALA), is reported here. After incubation with this molecule, tumor-targeted lymphocytes spontaneously synthesize anti-neoplastic drug protoporphyrin X (PpIX), and specifically accumulate in cancer tissue. Under periodic 630 nm laser irradiation, lymphocytes generate vesicle-like apoptotic body (Ab) containing the above-produced PpIX, and the facilitated delivery of PpIX from Ab makes an excellent therapeutic effect for Ras-mutated cancer cells under a second irradiation. Importantly, a microfluidic device is further fabricated to simplify cell sorting and drug synthesis with a one-step operation, which could promote generalization of this strategy. In vitro and in vivo studies confirm the success of such an easy-operated and global-regulated strategy for cancer therapy.

PMID: 29926990 [PubMed - as supplied by publisher]

[The protective effects of Astragaloside Ⅳ on diastolic function of rat thoracic aortic rings impaired by microvesicles].

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[The protective effects of Astragaloside Ⅳ on diastolic function of rat thoracic aortic rings impaired by microvesicles].

Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2018 Feb 08;34(2):164-168

Authors: Li YY, Shang M, Zhang KW, Wei S, Liu C, Zhu Q, Zhao JY, Wu YN, Song JQ, Liu YX

Abstract
OBJECTIVES: To investigate the effects of Astragaloside IV (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs), and the mechanism of AST.
METHODS: H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs in vitro under the condition of hypoxia for 12 hour/Reoxygenation for 4 hour, H/R-EMVs were stored in D-Hank's solution. Male Wistar rats were underwent thoracotomy, the thoracic aorta with intact endothelium were carefully removed and cut into 3~4 mm rings. The experiment was divided into six groups. H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10μg/ml; different doses of AST groups:thoracic aortic rings of rats were treated with 10, 20, 40, 60 mg/L AST co-incubated with 10μg/ml H/R-EMVs respectively; control group were treated with the same volume of D-Hank's solution. Duration of incubation was 4 h, each group was tested in five replicate aortic rings. Effects of AST on endothelium-dependent relaxation were detected. The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinase (Akt), phosphorylated Akt (p-Akt, Ser-473), extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2, Thr202/Tyr204) of rat thoracic aortic rings were detected.
RESULTS: Tenμg/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (P<0.01). Compared with H/R-EMVs group, relaxation of rat thoracic aortic rings was increased by 20, 40 and 60 mg/L AST in a concentration-dependent manner (P<0.01), the level of NO production was also enhanced (P<0.05, P<0.01). The level of t-eNOS, t-Akt and ERK1/2 was not changed, but the level of p-eNOS, p-Akt and p-ERK1/2 increased by the treatment with AST (P<0.01).
CONCLUSIONS: AST could effectively ameliorate endotheliumdependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner, the mechanism might involve the increase in production of NO, and the protein level of p-eNOS, p-Akt and p-ERK1/2.

PMID: 29926683 [PubMed - in process]

Mitochondrial Dysfunction in Stroke: Implications of Stem Cell Therapy.

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Mitochondrial Dysfunction in Stroke: Implications of Stem Cell Therapy.

Transl Stroke Res. 2018 Jun 20;:

Authors: Sarmah D, Kaur H, Saraf J, Vats K, Pravalika K, Wanve M, Kalia K, Borah A, Kumar A, Wang X, Yavagal DR, Dave KR, Bhattacharya P

Abstract
Stroke is a debilitating condition which is also the second leading cause of death and disability worldwide. Despite the benefits and promises shown by numerous neuroprotective agents in animal stroke models, their clinical translation has not been a complete success. Hence, search for treatment options have directed researchers towards utilising stem cells. Mitochondria has a major involvement in the pathophysiology of stroke and a number of other conditions. Stem cells have shown the ability to transfer mitochondria to the damaged cells and to help revive cell energetics in the recipient cell. The present review discusses how stem cells could be employed to protect neurons and mitochondria in stroke and also the various mechanisms involved in neuroprotection.

PMID: 29926383 [PubMed - as supplied by publisher]

Thermal carbonization in nanoscale reactors: controlled formation of carbon nanodots inside porous CaCO3 microparticles.

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Thermal carbonization in nanoscale reactors: controlled formation of carbon nanodots inside porous CaCO3 microparticles.

Sci Rep. 2018 Jun 20;8(1):9394

Authors: Vostrikova AV, Prikhozhdenko ES, Mayorova OA, Goryacheva IY, Tarakina NV, Sukhorukov GB, Sapelkin AV

Abstract
Synthesis of carbon nanodots (CNDs) in confined geometry via incorporation of dextran sulphate into pores of CaCO3 microparticles is demonstrated. The preparation process included three steps: co-precipitation of solutions of inorganic salts and carbon source, thermal treatment and CaCO3 matrix removal. We show that geometric constraints can be used to precisely control the amount of source material and to avoid formation of large carbon particles. Analysis of TEM data shows particle size of ~3.7 nm with narrow size distribution. Furthermore, we found that variation in pore morphology has a clear effect on CNDs structure and optical properties. CNDs with graphene oxide like structure were obtained in the nanoporous outer shell layer of CaCO3 microparticles, while less ordered CNDs with the evidence of complex disordered carbons were extracted from the inner microcavity. These results suggest that confined volume synthesis route in CaCO3 nanopores can be used to precisely control the structure and optical properties of CNDs.

PMID: 29925932 [PubMed - in process]

Label-free detection of hypoxia-induced extracellular vesicle secretion from MCF-7 cells.

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Label-free detection of hypoxia-induced extracellular vesicle secretion from MCF-7 cells.

Sci Rep. 2018 Jun 20;8(1):9402

Authors: Kilic T, Valinhas ATS, Wall I, Renaud P, Carrara S

Abstract
Nanoscale extracellular vesicles (EVs) including exosomes (50-150 nm membrane particles) have emerged as promising cancer biomarkers due to the carried genetic information about the parental cells. However the sensitive detection of these vesicles remains a challenge. Here we present a label-free electrochemical sensor to measure the EVs secretion levels of hypoxic and normoxic MCF-7 cells. The sensor design includes two consecutive steps; i) Au electrode surface functionalization for anti-CD81 Antibody and ii) EVs capture. The label-free detection of EVs was done via Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS). The working linear range for the sensor was 102-109 EVs/ml with an LOD 77 EVs/mL and 379 EVs/ml for EIS and DPV based detection. A blood-abundant protein, RhD was used for the selectivity test. In order to assess the performance of the biosensor, the level of EVs secretion by the human breast cancer MCF-7 cell line was compared with enzyme-linked immunosorbent assays (ELISA) and Nanoparticle Tracking Analysis (NTA). Designed label-free electrochemical sensors utilized for quantification of EVs secretion enhancement due to CoCl2-induced hypoxia and 1.23 fold increase with respect to normoxic conditions was found.

PMID: 29925885 [PubMed - in process]

H1.0 Linker Histone as an Epigenetic Regulator of Cell Proliferation and Differentiation.

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H1.0 Linker Histone as an Epigenetic Regulator of Cell Proliferation and Differentiation.

Genes (Basel). 2018 Jun 20;9(6):

Authors: Di Liegro CM, Schiera G, Di Liegro I

Abstract
H1 linker histones are a class of DNA-binding proteins involved in the formation of supra-nucleosomal chromatin higher order structures. Eleven non-allelic subtypes of H1 are known in mammals, seven of which are expressed in somatic cells, while four are germ cell-specific. Besides having a general structural role, H1 histones also have additional epigenetic functions related to DNA replication and repair, genome stability, and gene-specific expression regulation. Synthesis of the H1 subtypes is differentially regulated both in development and adult cells, thus suggesting that each protein has a more or less specific function. The somatic variant H1.0 is a linker histone that was recognized since long ago to be involved in cell differentiation. Moreover, it has been recently found to affect generation of epigenetic and functional intra-tumor heterogeneity. Interestingly, H1.0 or post-translational forms of it have been also found in extracellular vesicles (EVs) released from cancer cells in culture, thus suggesting that these cells may escape differentiation at least in part by discarding H1.0 through the EV route. In this review we will discuss the role of H1.0 in development, differentiation, and stem cell maintenance, also in relation with tumorigenesis, and EV production.

PMID: 29925815 [PubMed]

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

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Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

JCI Insight. 2018 Jun 21;3(12):

Authors: Merle NS, Grunenwald A, Rajaratnam H, Gnemmi V, Frimat M, Figueres ML, Knockaert S, Bouzekri S, Charue D, Noe R, Robe-Rybkine T, Le-Hoang M, Brinkman N, Gentinetta T, Edler M, Petrillo S, Tolosano E, Miescher S, Le Jeune S, Houillier P, Chauvet S, Rabant M, Dimitrov JD, Fremeaux-Bacchi V, Blanc-Brude OP, Roumenina LT

Abstract
In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

PMID: 29925688 [PubMed - as supplied by publisher]

Nanoparticle decoration impacts airborne fungal pathobiology.

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Nanoparticle decoration impacts airborne fungal pathobiology.

Proc Natl Acad Sci U S A. 2018 Jun 20;:

Authors: Westmeier D, Solouk-Saran D, Vallet C, Siemer S, Docter D, Götz H, Männ L, Hasenberg A, Hahlbrock A, Erler K, Reinhardt C, Schilling O, Becker S, Gunzer M, Hasenberg M, Knauer SK, Stauber RH

Abstract
Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

PMID: 29925597 [PubMed - as supplied by publisher]

[Role and mechanism of lipopolysaccharide induced exosome in the pathogenesis of acute lung injury].

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[Role and mechanism of lipopolysaccharide induced exosome in the pathogenesis of acute lung injury].

Zhonghua Yi Xue Za Zhi. 2018 Jun 12;98(22):1780-1785

Authors: Zheng BB, Zhang Y, Sun NN, Huang WH, Meng Y

Abstract
Objective: To explore the effect and mechanism of exosome derived from lipopolysaccharide (LPS)-induced mouse macrophage (RAW264.7) on acute lung injury. Methods: RAW264.7 were cultured in vitro and divided into 2 groups: control group and LPS-induced group. The exosomes were extracted from the two groups of cell supernatant by ultracentrifugation and classified into 2 groups: C-EXO group and LPS-EXO group. In vivo, random allocation was used to averagely divide the eighteen male C57BL/6 mice into 3 groups: control group, EXO-control group and EXO-LPS group. All mice were sacrificed after 12 h. The lung tissue was used for HE staining to assess the degree of acute lung injury as well as immunohistochemical staining for interleukin (IL) -1β and tumor necrosis factor (TNF)-α. The tissue protein expression levels of IL-1β, TNF-α, β-catenin, E-cadhein, ZO-1 and Occludin were measured by Western blot. In vitro, alveolar type Ⅱ epithelial cells (MLE-12) were cultured and divided into 3 groups: C-control group, EXO-control-induced group and EXO-LPS-induced group. The tissue protein expression levels of IL-1β, TNF-α, and Occludin were measured by Western blot after 12 h. Results: The two samples of C-EXO group and LPS-EXO group was proved to be exosomes. Under a light microscope, the lung tissue of EXO-LPS group showed inflammatory cell infiltration, hemorrhage, interstitial and alveolar edema, and the thickness of alveolar septum. The tissue protein levels of IL-1β and TNF-α in EXO-LPS group were obviously higher than the control group, EXO-control group (1.331±0.203 and 0.274±0.018, 0.892±0.074; 0.800±0.096 and 0.596±0.025, 0.441±0.061; all P<0.05). While the tissue protein levels of Occludin showed the opposite phenomenon (0.251±0.021 and 0.862±0.029, 0.453±0.013; all P<0.05). In vitro, Compared with the C-control group and the EXO-control-induced group, the expression levels of IL-1β and TNF-α increased significantly in the EXO-LPS-induced group (0.900±0.033 and 0.320±0.030, 0.661±0.028; 0.739±0.045 and 0.151±0.024, 0.360±0.037; all P<0.05). whereas the protein levels of Occludin expression were reversed in MLE-12 (0.585±0.082 and 0.941±0.090, 0.732±0.083; all P<0.05). Conclusion: Exosomes derived from LPS-induced RAW264.7 can induced the acute lung injury by affecting barrier function, which probably is related to the low degree of Occludin in alveolar type Ⅱ epithelial cells.

PMID: 29925159 [PubMed - in process]

Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease.

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Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease.

FASEB J. 2018 Jun 20;:fj201801001

Authors: Goetzl EJ, Nogueras-Ortiz C, Mustapic M, Mullins RJ, Abner EL, Schwartz JB, Kapogiannis D

Abstract
Exosomes derived from chondroitin sulfate proteoglycan (CSPG) 4 type neural precursor cells (CSPG4Es) were purified from human plasma by sequential immunoabsorption with anti-CSPG4 and anti-platelet growth factor receptor α mAb to characterize the potential in vivo roles of CSPG4 cells in neuronal repair. Hepatocyte growth factor, fibroblast growth factors (FGFs)-2 and -13, and type 1 insulin-like growth factor (IGF-1), which enhance neuronal survival and functions, were quantified in CSPG4E extracts. For CSPG4Es of 24 healthy control subjects, mean levels of hepatocyte growth factor, FGF-13, and IGF-1, but not FGF-2, were significantly higher by up to 7-fold than in their neuronal-derived exosomes, and mean levels of all 4 growth factors were significantly higher by up to 8-fold than in their astrocyte-derived exosomes. Mean CSPG4E levels of all growth factors were significantly lower in patients with mild Alzheimer disease (AD) ( n = 24) than in age- and sex-matched cognitively normal control subjects ( n = 24). Mean CSPG4E levels of all growth factors were also significantly lower in 15 patients at the stage of moderate dementia from AD (AD2) and at their preclinical stage 3 to 8 yr earlier (AD1), with no differences between values at stages AD1 and AD2. Current findings suggest that CSPG4 cells export in exosomes higher levels of neurotrophic factors than neurons or astrocytes and that CSPG4E neurotrophic factors are diminished early in AD, with no significant progression of decreases later in the course.-Goetzl, E. J., Nogueras-Ortiz, C., Mustapic, M., Mullins, R. J., Abner, E. L., Schwartz, J. B., Kapogiannis, D. Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease.

PMID: 29924942 [PubMed - as supplied by publisher]

TCTP in Extracellular Vehicles: Dangerous Cargo?

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TCTP in Extracellular Vehicles: Dangerous Cargo?

Am J Respir Cell Mol Biol. 2018 Jun 20;:

Authors: Jandl K, Gregory CD, Kwapiszewska G

PMID: 29924940 [PubMed - as supplied by publisher]

Effective refractive index and lipid content of extracellular vesicles revealed using optical waveguide scattering and fluorescence microscopy.

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Effective refractive index and lipid content of extracellular vesicles revealed using optical waveguide scattering and fluorescence microscopy.

Langmuir. 2018 Jun 20;:

Authors: Rupert DLM, Mapar M, Shelke GV, Norling K, Elmeskog M, Lotvall JO, Block S, Bally M, Agnarsson B, Höök F

Abstract
Extracellular vesicles (EVs) are generating a growing interest due to the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different sub-populations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Due to the large heterogeneity of EV samples, such information remains rather elusive and there is accordingly a need for new and complementary characterization schemes that can help expanding the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were pre-separated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high density (HD) population was on average a factor of 3 lower than that of the low density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single particle level. The labelled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index (n ~ 1.35) compared with the LD EVs (n ~1.38), indicating that both the lipid and protein content was indeed lower in the HD EVs. While separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs; i.e. the two gradient bands are more likely due to differences in membrane permeability than to differences in biomolecular content of the EVs.

PMID: 29923735 [PubMed - as supplied by publisher]

In Situ Depot for Continuous Evolution of Gaseous H2 Mediated by Magnesium Passivation/Activation Cycle for Treating Osteoarthritis.

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In Situ Depot for Continuous Evolution of Gaseous H2 Mediated by Magnesium Passivation/Activation Cycle for Treating Osteoarthritis.

Angew Chem Int Ed Engl. 2018 Jun 20;:

Authors: Wan WL, Lin YJ, Shih PC, Bow YR, Cui Q, Chang Y, Chia WT, Sung HW

Abstract
Inflammation is involved in many human pathologies, including osteoarthritis (OA). Hydrogen (H2) is known to have anti-inflammatory effects; however, the bioavailability of directly administered H2 gas is typically poor. Herein, a local delivery system that can provide a high therapeutic concentration of gaseous H2 at inflamed tissues is proposed. The delivery system comprises poly(lactic-co-glycolic acid) microparticles that contain magnesium powder (Mg@PLGA MPs). Mg@PLGA MPs that are intra-muscularly injected close to the OA knee in a mouse model can act as an in situ depot that can evolve gaseous H2 continuously, mediated by the cycle of passivation/activation of Mg in body fluids, at a concentration that exceeds its therapeutic threshold. The analytical data that are obtained in the biochemical and histological studies indicate that the proposed Mg@PLGA MPs can effectively mitigate tissue inflammation and prevent cartilage from destruction, arresting the progression of OA changes.

PMID: 29923670 [PubMed - as supplied by publisher]

Extracellular vesicles and microvascular pathology: decoding the active dialogue.

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Extracellular vesicles and microvascular pathology: decoding the active dialogue.

Microcirculation. 2018 Jun 20;:e12485

Authors: Beheshti EH, Grau GER

Abstract
More than seventy years ago Chargaff and West reported the presence of platelet-derived particles in normal plasma. Since then, an enormous number of studies have aimed at characterising the diversity of extracellular vesicles (EV) and understanding their role in biological and pathological processes, both as elements of communication vesicles and as biological transporters. This article is protected by copyright. All rights reserved.

PMID: 29923276 [PubMed - as supplied by publisher]

Harnessing extracellular vesicles to direct endochondral repair of large bone defects.

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Harnessing extracellular vesicles to direct endochondral repair of large bone defects.

Bone Joint Res. 2018 Apr;7(4):263-273

Authors: Ferreira E, Porter RM

Abstract
Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation. Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263-273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.

PMID: 29922444 [PubMed]

Vesicle-Mediated Control of Cell Function: The Role of Extracellular Matrix and Microenvironment.

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Vesicle-Mediated Control of Cell Function: The Role of Extracellular Matrix and Microenvironment.

Front Physiol. 2018;9:651

Authors: Rackov G, Garcia-Romero N, Esteban-Rubio S, Carrión-Navarro J, Belda-Iniesta C, Ayuso-Sacido A

Abstract
Extracellular vesicles (EVs) - including exosomes, microvesicles and apoptotic bodies - have received much scientific attention last decade as mediators of a newly discovered cell-to-cell communication system, acting at short and long distances. EVs carry biologically active molecules, thus providing signals that influence a spectrum of functions in recipient cells during various physiological and pathological processes. Recent findings point to EVs as very attractive immunomodulatory therapeutic agents, vehicles for drug delivery and diagnostic and prognostic biomarkers in liquid biopsies. In addition, EVs interact with and regulate the synthesis of extracellular matrix (ECM) components, which is crucial for organ development and wound healing, as well as bone and cardiovascular calcification. EVs carrying matrix metalloproteinases (MMPs) are involved in ECM remodeling, thus modifying tumor microenvironment and contributing to premetastatic niche formation and angiogenesis. Here we review the role of EVs in control of cell function, with emphasis on their interaction with ECM and microenvironment in health and disease.

PMID: 29922170 [PubMed]

Fatty Acids and Calcium Regulation in Prostate Cancer.

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Fatty Acids and Calcium Regulation in Prostate Cancer.

Nutrients. 2018 Jun 19;10(6):

Authors: Maly IV, Hofmann WA

Abstract
Prostate cancer is a widespread malignancy characterized by a comparative ease of primary diagnosis and difficulty in choosing the individualized course of treatment. Management of prostate cancer would benefit from a clearer understanding of the molecular mechanisms behind the transition to the lethal, late-stage forms of the disease, which could potentially yield new biomarkers for differential prognosis and treatment prioritization in addition to possible new therapeutic targets. Epidemiological research has uncovered a significant correlation of prostate cancer incidence and progression with the intake (and often co-intake) of fatty acids and calcium. Additionally, there is evidence of the impact of these nutrients on intracellular signaling, including the mechanisms mediated by the calcium ion as a second messenger. The present review surveys the recent literature on the molecular mechanisms associated with the critical steps in the prostate cancer progression, with special attention paid to the regulation of these processes by fatty acids and calcium homeostasis. Testable hypotheses are put forward that integrate some of the recent results in a more unified picture of these phenomena at the interface of cell signaling and metabolism.

PMID: 29921791 [PubMed - in process]

Dynamic Cultivation of Mesenchymal Stem Cell Aggregates.

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Dynamic Cultivation of Mesenchymal Stem Cell Aggregates.

Bioengineering (Basel). 2018 Jun 19;5(2):

Authors: Egger D, Tripisciano C, Weber V, Dominici M, Kasper C

Abstract
Mesenchymal stem cells (MSCs) are considered as primary candidates for cell-based therapies due to their multiple effects in regenerative medicine. Pre-conditioning of MSCs under physiological conditions&mdash;such as hypoxia, three-dimensional environments, and dynamic cultivation&mdash;prior to transplantation proved to optimize their therapeutic efficiency. When cultivated as three-dimensional aggregates or spheroids, MSCs display increased angiogenic, anti-inflammatory, and immunomodulatory effects as well as improved stemness and survival rates after transplantation, and cultivation under dynamic conditions can increase their viability, proliferation, and paracrine effects, alike. Only few studies reported to date, however, have utilized dynamic conditions for three-dimensional aggregate cultivation of MSCs. Still, the integration of dynamic bioreactor systems, such as spinner flasks or stirred tank reactors might pave the way for a robust, scalable bulk expansion of MSC aggregates or MSC-derived extracellular vesicles. This review summarizes recent insights into the therapeutic potential of MSC aggregate cultivation and focuses on dynamic generation and cultivation techniques of MSC aggregates.

PMID: 29921755 [PubMed]

Innovative Technologies Changing Cancer Treatment.

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Innovative Technologies Changing Cancer Treatment.

Cancers (Basel). 2018 Jun 19;10(6):

Authors: Charmsaz S, Prencipe M, Kiely M, Pidgeon GP, Collins DM

Abstract
Conventional therapies for cancer such as chemotherapy and radiotherapy remain a mainstay in treatment, but in many cases a targeted approach is lacking, and patients can be vulnerable to drug resistance. In recent years, novel concepts have been emerging to improve the traditional therapeutic options in cancers with poor survival outcomes. New therapeutic strategies involving areas like energy metabolism and extracellular vesicles along with advances in immunotherapy and nanotechnology are driving the next generation of cancer treatments. The development of fields such as theranostics in nanomedicine is also opening new doors for targeted drug delivery and nano-imaging. Here we discuss the use of innovative technologies presented at the Irish Association for Cancer Research (IACR) Annual Meeting, highlighting examples of where new approaches may lead to promising new treatment options for a range of cancer types.

PMID: 29921753 [PubMed]

Tumor-Educated Platelets as a Noninvasive Biomarker Source for Cancer Detection and Progression Monitoring.

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Tumor-Educated Platelets as a Noninvasive Biomarker Source for Cancer Detection and Progression Monitoring.

Cancer Res. 2018 Jun 19;:

Authors: Best MG, Wesseling P, Wurdinger T

Abstract
Liquid biopsies represent a potential revolution in cancer diagnostics as a noninvasive method for detecting and monitoring diseases, complementary to or even replacing current tissue biopsy approaches. Several blood-based biosources and biomolecules, such as cell-free DNA and RNA, proteins, circulating tumor cells, and extracellular vesicles, have been explored for molecular test development. We recently discovered the potential of tumor-educated blood platelets (TEP) as a noninvasive biomarker trove for RNA biomarker panels. TEPs are involved in the progression and spread of several solid tumors, and spliced TEP RNA surrogate signatures can provide specific information on the presence, location, and molecular characteristics of cancers. So far, TEP samples from patients with different tumor types, including lung, brain, and breast cancers, have been tested, and it has been shown that TEPs from patients with cancer are distinct from those with inflammatory and other noncancerous diseases. It remains to be investigated how platelets are "educated," which mechanisms cause intraplatelet RNA splicing, and whether the relative contribution of specific platelet subpopulations changes in patients with cancer. Ultimately, TEP RNA may complement currently used biosources and biomolecules employed for liquid biopsy diagnosis, potentially enhancing the detection of cancer in an early stage and facilitating noninvasive disease monitoring. Cancer Res; 78(13); 1-6. ©2018 AACR.

PMID: 29921699 [PubMed - as supplied by publisher]

Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of mir-125b.

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Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of mir-125b.

Circ Res. 2018 Jun 19;:

Authors: Xiao C, Wang K, Xu Y, Hu H, Zhang N, Wang Y, Zhong Z, Zhao J, Li Q, Zhu D, Ke C, Zhong S, Wu X, Yu H, Zhu W, Chen J, Zhang J, Wang J, Hu X

Abstract
Rationale: Autophagy can preserve cell viability under conditions of mild ischemic stress by degrading damaged organelles for ATP production, but under conditions of severe ischemia, it can promote cell death and worsen cardiac performance. Mesenchymal stem cells (MSCs) are cardioprotective when tested in animal models of myocardial infarction (MI), but whether these benefits occur through the regulation of autophagy is unknown. Objective: To determine whether transplanted MSCs reduce the rate of autophagic degradation (autophagic flux) in infarcted hearts and if so, to characterize the mechanisms involved. Methods and Results: Treatment with transplanted MSCs improved cardiac function and infarct size while reducing apoptosis and measures of autophagic flux (BafA1-induced LC3-II accumulation and autophagosome/autolysosome prevalence) in infarcted mouse hearts. In hypoxia and serum deprivation (H/SD)-cultured neonatal mouse cardiomyocytes (NMCMs), autophagic flux and cell death, as well as p53-Bnip3 signaling, declined when the cells were cultured with MSCs or MSCs-secreted exosomes, but the changes associated with MSCs-secreted exosomes (MSCs-exo) were largely abolished by pretreatment with the exosomal inhibitor GW4869. Furthermore, a mimic of the exosomal oligonucleotide miR-125b reduced, while an anti-miR-125b oligonucleotide increased, autophagic flux, cell death, via modulating p53-Bnip3 signaling in H/SD-cultured NMCMs. In the in vivo mouse MI model, MSCs-exo, but not the exosomes obtained from MSCs pretreated with the anti-miR-125b oligonucleotide (MSCs-exoanti-miR-125b), recapitulated the same results as the in vitro experiments. Moreover, measurements of infarct size and cardiac function were significantly better in group that were treated with MSCs-exo than the MSCs-exoanti-miR-125b group. Conclusions: The beneficial effects offered by MSCs transplantation after MI are at least partially due to improved autophagic flux through excreted exosome containing mainly miR-125b-5p.

PMID: 29921652 [PubMed - as supplied by publisher]

Exosomes of glioma cells deliver miR-148a to promote proliferation and metastasis of glioblastoma via targeting CADM1miR-148a effect on glioma cells by CADM1.

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Exosomes of glioma cells deliver miR-148a to promote proliferation and metastasis of glioblastoma via targeting CADM1miR-148a effect on glioma cells by CADM1.

Bull Cancer. 2018 Jun 16;:

Authors: Cai Q, Zhu A, Gong L

Abstract
Exosomes are now considered to be involved in mediating cell-to-cell communication to promote or inhibit tumor progression. However, the role and molecular mechanism of exosomes in promoting glioblastoma (GBM) metastasis remains elusive. Here, we found that circulating exosomal miR-148a levels were significantly higher in serum from GBM patients compared with serum from healthy volunteers. In T98G cells, inhibition of miR-148a suppressed cell proliferation and metastasis. In addition, we identified Cell adhesion molecule 1 (CADM1) as a target gene of miR-148a using luciferase reporter assay. Both protein and mRNA levels of CADM1 were decreased in tissues from GBM patients. There was a strong negative correlation between exosomal miR-148a and CADM1 mRNA levels in samples of patients. Moreover, miR-148a antagonist increased p-STAT3 protein level to activate STAT3 pathway. In conclusion, our findings indicated that miR-148a delivered by exosomes may promote cancer cell proliferation and metastasis via targeting CADM1 to activate STAT3 pathway, suggesting a predictor and therapeutic target role of exosomal miR-148a in GBM patients.

PMID: 29921422 [PubMed - as supplied by publisher]

Inhibition of the RhoGTPase Cdc42 by ML141 enhances hepatocyte differentiation from human adipose-derived mesenchymal stem cells via the Wnt5a/PI3K/miR-122 pathway: impact of the age of the donor.

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Inhibition of the RhoGTPase Cdc42 by ML141 enhances hepatocyte differentiation from human adipose-derived mesenchymal stem cells via the Wnt5a/PI3K/miR-122 pathway: impact of the age of the donor.

Stem Cell Res Ther. 2018 Jun 19;9(1):167

Authors: Chaker D, Mouawad C, Azar A, Quilliot D, Achkar I, Fajloun Z, Makdissy N

Abstract
BACKGROUND: Human adipose-derived mesenchymal stem cells (hADSCs) are promising cells that may promote hepatocyte differentiation (Hep-Dif) and improve liver function, but the involvement of Cdc42, a key small RhoGTPase which plays a crucial role in aging, is still not well established. We hypothesized that the inhibition of Cdc42 may rescue the hepatogenic potential of hADSCs derived from aged donors.
METHODS: hADSCs isolated from 61 women of different ages were cultured for evaluation of the proliferation of cells, adherence, apoptosis, immunomodulation, immunophenotyping, multipotency, gene expression, and cell function during Hep-Dif. Inhibition of Cdc42 by ML141 was realized during two phases: initiation (days -2 to 14 (D-2/14)) from undifferentiated to hepatoblast-like cells, or maturation (days 14 to 28 (D14/28)) from undifferentiated to hepatocyte-like cells. Mechanistic insights of the Wnt(s)/MAPK/PI3K/miR-122 pathways were studied.
RESULTS: Cdc42 activity in undifferentiated hADSCs showed an age-dependent significant increase in Cdc42-GTP correlated to a decrease in Cdc42GAP; the low potentials of cell proliferation, doubling, adherence, and immunomodulatory ability (proinflammatory over anti-inflammatory) contrary to the apoptotic index of the aged group were significantly reversed by ML141. Aged donor cells showed a decreased potential for Hep-Dif which was rescued by ML141 treatment, giving rise to mature and functional hepatocyte-like cells as assessed by hepatic gene expression, cytochrome activity, urea and albumin production, low-density lipoprotein (LDL) uptake, and glycogen storage. ML141-induced Hep-Dif showed an improvement in mesenchymal-epithelial transition, a switch from Wtn-3a/β-catenin to Wnt5a signaling, involvement of PI3K/PKB but not the MAPK (ERK/JNK/p38) pathway, induction of miR-122 expression, reinforcing the exosomes release and the production of albumin, and epigenetic changes. Inhibition of PI3K and miR-122 abolished completely the effects of ML141 indicating that inhibition of Cdc42 promotes the Hep-Dif through a Wnt5a/PI3K/miR-122/HNF4α/albumin/E-cadherin-positive action. The ML141(D-2/14) protocol had more pronounced effects when compared with ML141(D14/28); inhibition of DNA methylation in combination with ML141(D-2/14) showed more efficacy in rescuing the Hep-Dif of aged hADSCs. In addition to Hep-Dif, the multipotency of aged hADSC-treated ML141 was observed by rescuing the adipocyte and neural differentiation by inducing PPARγ/FABP4 and NeuN/O4 but inhibiting Pref-1 and GFAP, respectively.
CONCLUSION: ML141 has the potential to reverse the age-related aberrations in aged stem cells and promotes their hepatogenic differentiation. Selective inhibition of Cdc42 could be a potential target of drug therapy for aging and may give new insights on the improvement of Hep-Dif.

PMID: 29921325 [PubMed - in process]

Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes.

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Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes.

Parasit Vectors. 2018 Jun 19;11(1):355

Authors: Marshall S, Kelly PH, Singh BK, Pope RM, Kim P, Zhanbolat B, Wilson ME, Yao C

Abstract
BACKGROUND: The Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.
METHODS: Using mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.
RESULTS: Metacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.
CONCLUSIONS: The data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

PMID: 29921321 [PubMed - in process]

COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal.

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COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal.

Elife. 2017 Oct 23;6:

Authors: Xu P, Hankins HM, MacDonald C, Erlinger SJ, Frazier MN, Diab NS, Piper RC, Jackson LP, MacGurn JA, Graham TR

Abstract
The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway.

PMID: 29058666 [PubMed - indexed for MEDLINE]

Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection.

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Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection.

Inflammopharmacology. 2017 Dec;25(6):643-651

Authors: Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM

Abstract
Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected.

PMID: 28528362 [PubMed - indexed for MEDLINE]

 

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