Publications by authors named "Bernd Giebel"

94 Publications

Analysis of individual extracellular vesicles by imaging flow cytometry.

Methods Enzymol 2020 22;645:55-78. Epub 2020 Jun 22.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Virtually all cells release extracellular vesicles (EVs) into their environment, such as exosomes and microvesicles. EVs can mediate intercellular communication processes in a targeted manner. Representing their cell of origin, EVs contain cell type specific signatures, qualifying them as a novel class of biomarkers. Furthermore, according to their tropism to certain target cells, EVs provide promising aspects to be used as drug delivery vehicles. Depending on their origin, certain EVs contain the potential to modulate physiological and pathophysiological processes. Although the EV field provides many interesting aspects, the methodology in EV research is limited. For now, EVs are mainly analyzed by nanoparticle tracking analysis and bulk molecular analysis, regularly Western Blot. These technologies cannot dissect the heterogeneity of EVs observed by electron microscopy (EM). Although EM technologies help to demonstrate the heterogeneity within EV samples, EM technologies are not appropriate to perform more complex and quantitative EV analyses. Flow cytometry (FCM) is a traditional method for dissecting the heterogeneity of given cell populations in a quantitative and complex manner. However, classical FCM regularly fails to detect objects in the size range of small EVs (sEVs) that typically is in the range between 70 and 150nm. Recently, we and others demonstrated the potential of imaging FCM for the analyses of small EVs at the single vesicle level. Here, at the example of sEVs harvested from supernatants of human mesenchymal stromal cells (MSCs), we share a protocol for studying the expression of the tetraspanins CD9, CD63 and CD81 on single EVs.
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http://dx.doi.org/10.1016/bs.mie.2020.05.013DOI Listing
June 2020

Circulating MicroRNAs: Posttranscriptional Regulators and Disease Markers Holding Promise in Stroke Prediction.

Stroke 2021 Mar 10;52(3):954-956. Epub 2021 Feb 10.

Institute of Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Germany (B.G.).

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http://dx.doi.org/10.1161/STROKEAHA.120.033688DOI Listing
March 2021

Perinatal Derivatives: Where Do We Stand? A Roadmap of the Human Placenta and Consensus for Tissue and Cell Nomenclature.

Front Bioeng Biotechnol 2020 17;8:610544. Epub 2020 Dec 17.

Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy.

Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and characterization and clinical application of PnD.
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http://dx.doi.org/10.3389/fbioe.2020.610544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773933PMC
December 2020

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Reduce Neuroinflammation, Promote Neural Cell Proliferation and Improve Oligodendrocyte Maturation in Neonatal Hypoxic-Ischemic Brain Injury.

Front Cell Neurosci 2020 10;14:601176. Epub 2020 Dec 10.

Department of Pediatrics I, Neonatology and Experimental Perinatal Neurosciences, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of childhood mortality and disability. Stem cell-based regenerative therapies seem promising to prevent long-term neurological deficits. Our previous work in neonatal HI revealed an unexpected interaction between mesenchymal stem/stromal cells (MSCs) and the brains' microenvironment leading to an altered therapeutic efficiency. MSCs are supposed to mediate most of their therapeutic effects in a paracrine mode via extracellular vesicles (EVs), which might be an alternative to cell therapy. In the present study, we investigated the impact of MSC-EVs on neonatal HI-induced brain injury. Nine-day-old C57BL/6 mice were exposed to HI through ligation of the right common carotid artery followed by 1 h hypoxia (10% oxygen). MSC-EVs were injected intraperitoneally 1, 3, and 5 days after HI. One week after HI, brain injury was evaluated by regional neuropathological scoring, atrophy measurements and immunohistochemistry to assess effects on neuronal, oligodendrocyte and vessel densities, proliferation, oligodendrocyte maturation, myelination, astro-, and microglia activation. Immunohistochemistry analyses were complemented by mRNA expression analyses for a broad set of M1/M2- and A1/A2-associated molecules and neural growth factors. While total neuropathological scores and tissue atrophy were not changed, MSC-EVs significantly protected from HI-induced striatal tissue loss and decreased micro- and astroglia activation. MSC-EVs lead to a significant downregulation of the pro-inflammatory cytokine TNFa, accompanied by a significant upregulation of the M2 marker YM-1 and the anti-inflammatory cytokine TGFb. MSC-EVs significantly decreased astrocytic expression of the A1 marker C3, concomitant with an increased expression of neural growth factors (i.e., BDNF, VEGF, and EGF). These alterations were associated with an increased neuronal and vessel density, coinciding with a significant increase of proliferating cells in the neurogenic sub-ventricular zone juxtaposed to the striatum. MSC-EV-mediated neuroprotection went along with a significant improvement of oligodendrocyte maturation and myelination. The present study demonstrates that MSC-EVs mediate anti-inflammatory effects, promote regenerative responses and improve key developmental processes in the injured neonatal brain. The present results suggest different cellular target mechanisms of MSC-EVs, preventing secondary HI-induced brain injury. MSC-EV treatment may be a promising alternative to risk-associated cell therapies in neonatal brain injury.
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http://dx.doi.org/10.3389/fncel.2020.601176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758466PMC
December 2020

Weiss Response to Sengupta et al. (DOI: 10.1089/scd.2020.0095).

Stem Cells Dev 2020 12;29(24):1533-1534

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

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http://dx.doi.org/10.1089/scd.2020.0114DOI Listing
December 2020

Acute myeloid leukemia-induced remodeling of the human bone marrow niche predicts clinical outcome.

Blood Adv 2020 Oct;4(20):5257-5268

Department of Hematology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Murine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However, little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here, we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly, their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact-dependent manner through inhibition of cytoplasmic β-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30% lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore, patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7% whereas patients with higher osteocalcin levels reached a survival rate of 66.8%. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients.
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http://dx.doi.org/10.1182/bloodadvances.2020001808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594397PMC
October 2020

Scaled Isolation of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles.

Curr Protoc Stem Cell Biol 2020 12;55(1):e128

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Mesenchymal stem/stromal cells (MSCs) provide therapeutic effects in many diseases. Contrary to initial hypotheses, they act in a paracrine rather than a cellular manner. To this end, extracellular vesicles (EVs) have been found to mediate the therapeutic effects, even when harvested from MSC-conditioned cell culture supernatants. Lacking self-replicating activity and being so small that MSC-EV preparations can be sterilized by filtration, EVs provide several advantages as therapeutic agents over cellular therapeutics. At present, methods allowing EV preparation from larger volumes are scarce and regularly require special equipment. We have developed a polyethylene glycol-based precipitation protocol allowing extraction of EVs from several liters of conditioned medium. MSC-EVs prepared with this method have been successfully applied to a human graft-versus-host disease patient and to several animal models. Although the method comes with its own limitations, it is extremely helpful for the initial evaluation of EV-based therapeutic approaches. Here, we introduce the technique in detail and discuss all critical steps. © 2020 The Authors. Basic Protocol 1: Preparation of MSC-conditioned medium for scaled MSC-EV production Basic Protocol 2: PEG precipitation OF MSC-EV from MSC-conditioned medium.
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http://dx.doi.org/10.1002/cpsc.128DOI Listing
December 2020

Exposure of Patient-Derived Mesenchymal Stromal Cells to TGFB1 Supports Fibrosis Induction in a Pediatric Acute Megakaryoblastic Leukemia Model.

Mol Cancer Res 2020 10 8;18(10):1603-1612. Epub 2020 Jul 8.

Department of Pediatric Hematology and Oncology, University Children's Hospital Essen, Essen, Germany.

Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL ( = 5) and ML-DS ( = 9). Healthy donor control MSCs ( = 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdc Il2rg/SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future. IMPLICATIONS: TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/10/1603/F1.large.jpg.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0091DOI Listing
October 2020

Re: "Exosomes Derived from Bone Marrow Mesenchymal Stem Cells as Treatment for Severe COVID-19" by Sengupta et al.

Stem Cells Dev 2020 07 10;29(14):877-878. Epub 2020 Jun 10.

GMP Unit, Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), University Hospital, Salzburg, Austria.

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http://dx.doi.org/10.1089/scd.2020.0089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374615PMC
July 2020

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

Cytotherapy 2020 09 16;22(9):482-485. Epub 2020 May 16.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. Electronic address:

Statement: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.
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http://dx.doi.org/10.1016/j.jcyt.2020.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229942PMC
September 2020

High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.

Cytometry A 2020 06 16;97(6):602-609. Epub 2020 May 16.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50-150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)-labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence-conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application-specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.24034DOI Listing
June 2020

Ultrasmall gold nanoparticles (2 nm) can penetrate and enter cell nuclei in an in vitro 3D brain spheroid model.

Acta Biomater 2020 07 13;111:349-362. Epub 2020 May 13.

Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Essen, 45117, Germany. Electronic address:

The neurovascular unit (NVU) is a complex functional and anatomical structure composed of endothelial cells and their blood-brain barrier (BBB) forming tight junctions. It represents an efficient barrier for molecules and drugs. However, it also prevents a targeted transport for the treatment of cerebral diseases. The uptake of ultrasmall nanoparticles as potential drug delivery agents was studied in a three-dimensional co-culture cell model (3D spheroid) composed of primary human cells (astrocytes, pericytes, endothelial cells). Multicellular 3D spheroids show reproducible NVU features and functions. The spheroid core is composed mainly of astrocytes, covered with pericytes, while brain endothelial cells form the surface layer, establishing the NVU that regulates the transport of molecules. After 120 h cultivation, the cells self-assemble into a 350 µm spheroid as shown by confocal laser scanning microscopy. The passage of different types of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm) both into the spheroid and into three constituting cell types was studied by confocal laser scanning microscopy. Three kinds of covalently fluorophore-conjugated gold nanoparticles were used: One with fluorescein (FAM), one with Cy3, and one with the peptide CGGpTPAAK-5,6-FAM-NH. In 2D cell co-culture experiments, it was found that all three kinds of nanoparticles readily entered all three cell types. FAM- and Cy3-labelled nanoparticles were able to enter the cell nucleus as well. The three dissolved dyes alone were not taken up by any cell type. A similar situation evolved with 3D spheroids: The three kinds of nanoparticles entered the spheroid, but the dissolved dyes did not. The presence of a functional blood-brain barrier was demonstrated by adding histamine to the spheroids. In that case, the blood-brain barrier opened, and dissolved dyes like a FITC-labelled antibody and FITC alone entered the spheroid. In summary, our results qualify ultrasmall gold nanoparticles as suitable carriers for imaging or drug delivery into brain cells (sometimes including the nucleus), brain cell spheroids, and probably also into the brain. STATEMENT OF SIGNIFICANCE: 3D brain spheroid model and its permeability by ultrasmall gold nanoparticles. We demonstrate that ultrasmall gold nanoparticles can easily penetrate the constituting cells and sometimes even enter the cell nucleus. They can also enter the interior of the blood-brain barrier model. In contrast, small molecules like fluorescing dyes are not able to do that. Thus, ultrasmall gold nanoparticles can serve as carriers of drugs or for imaging inside the brain.
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http://dx.doi.org/10.1016/j.actbio.2020.04.023DOI Listing
July 2020

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Stroke 2020 06 21;51(6):1825-1834. Epub 2020 Apr 21.

From the Department of Neurology (C.W., M.S., J.S., R.P., N.H., E.D., J.G., C.K., D.M.H.), University Hospital Essen, Germany.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.
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http://dx.doi.org/10.1161/STROKEAHA.119.028012DOI Listing
June 2020

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis.

J Cell Biol 2020 03;219(3)

Centre for Organismal Studies, University of Heidelberg, Heidelberg, Germany.

Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.
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http://dx.doi.org/10.1083/jcb.201907136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055001PMC
March 2020

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

J Extracell Vesicles 2020 3;9(1):1713526. Epub 2020 Feb 3.

Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1713526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034442PMC
February 2020

Extracellular vesicles isolated from patients undergoing remote ischemic preconditioning decrease hypoxia-evoked apoptosis of cardiomyoblasts after isoflurane but not propofol exposure.

PLoS One 2020 14;15(2):e0228948. Epub 2020 Feb 14.

Klinik für Anästhesiologie und Intensivmedizin, Universität Duisburg-Essen & Universitätsklinikum Essen, Essen, Germany.

Remote ischemic preconditioning (RIPC) can evoke cardioprotection following ischemia/reperfusion and this may depend on the anesthetic used. We tested whether 1) extracellular vesicles (EVs) isolated from humans undergoing RIPC protect cardiomyoblasts against hypoxia-induced apoptosis and 2) this effect is altered by cardiomyoblast exposure to isoflurane or propofol. EVs were isolated before and 60 min after RIPC or Sham from ten patients undergoing coronary artery bypass graft surgery with isoflurane anesthesia and quantified by Nanoparticle Tracking Analysis. Following EV-treatment for 6 hours under exposure of isoflurane or propofol, rat H9c2 cardiomyoblasts were cultured for 18 hours in normoxic or hypoxic atmospheres. Apoptosis was detected by flow cytometry. Serum nanoparticle concentrations in patients had increased sixty minutes after RIPC compared to Sham (2.5x1011±4.9x1010 nanoparticles/ml; Sham: 1.2x1011±2.0x1010; p = 0.04). Hypoxia increased apoptosis of H9c2 cells (hypoxia: 8.4%±0.6; normoxia: 2.5%±0.1; p<0.0001). RIPC-EVs decreased H9c2 cell apoptosis compared to control (apoptotic ratio: 0.83; p = 0.0429) while Sham-EVs showed no protection (apoptotic ratio: 0.97). Prior isoflurane exposure in vitro even increased protection (RIPC-EVs/control, apoptotic ratio: 0.79; p = 0.0035; Sham-EVs/control, apoptotic ratio:1.04) while propofol (50μM) abrogated protection by RIPC-EVs (RIPC-EVs/control, Apoptotic ratio: 1.01; Sham-EVs/control, apoptotic ratio: 0.94; p = 0.602). Thus, EVs isolated from patients undergoing RIPC under isoflurane anesthesia protect H9c2 cardiomyoblasts against hypoxia-evoked apoptosis and this effect is abrogated by propofol. This supports a role of human RIPC-generated EVs in cardioprotection and underlines propofol as a possible confounder in RIPC-signaling mediated by EVs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228948PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021285PMC
May 2020

CpG stimulation of chronic lymphocytic leukemia cells induces a polarized cell shape and promotes migration in vitro and in vivo.

PLoS One 2020 10;15(2):e0228674. Epub 2020 Feb 10.

Department of Hematology, University Hospital, University of Duisburg-Essen, Essen, Germany.

In order to accomplish their physiological functions leukocytes have the capability to migrate. As a prerequisite they need to adopt a polarized cell shape, forming a leading edge at the front and a uropod at rear pole. In this study we explored the capability of chronic lymphocytic leukaemia (CLL) cells to adopt this leukocyte-specific migration phenotype. Furthermore, we studied the impact of the Toll-like receptor 9 (TLR9) agonists CpGs type A, B and C and the antagonist oligodesoxynucleotide (ODN) INH-18 on the cell polarization and migration process of primary human CLL cells. Upon cultivation, a portion of purified CLL cells adopted polarized cell shapes spontaneously (range 10-38%). Stimulation with CpG ODNs type B (ODN 2006) and CpGs type C (ODN 2395) significantly increased the frequency of morphologically polarized CLL cells, while ODN INH-18 was hardly able to act antagonistically. Like in human hematopoietic stem and progenitor cells, in morphologically polarized CLL cells CXCR4 was redistributed to the leading edge and CD50 to the uropod. Coupled to the increased frequencies of morphologically polarized cells, CpGs type B and C stimulated CLL cells showed higher migration activities in vitro and following intravenous injection higher homing frequencies to the bone marrow of immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Thus, presumably independent of TLR-9 signaling, CpGs type B and C promote the cellular polarization process of CLL cells and their ability to migrate in vitro and in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228674PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010256PMC
May 2020

Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics.

Ann Hematol 2020 Mar 13;99(3):459-475. Epub 2020 Jan 13.

Department of Pediatric Hematology and Oncology, University Children's Hospital, University Hospital of Essen, Hufelandstrasse 55, 45147, Essen, Germany.

Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.
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http://dx.doi.org/10.1007/s00277-019-03866-wDOI Listing
March 2020

Human multipotent hematopoietic progenitor cell expansion is neither supported in endothelial and endothelial/mesenchymal co-cultures nor in NSG mice.

Sci Rep 2019 09 9;9(1):12914. Epub 2019 Sep 9.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Endothelial and mesenchymal stromal cells (ECs/MSCs) are crucial components of hematopoietic bone marrow stem cell niches. Both cell types appear to be required to support the maintenance and expansion of multipotent hematopoietic cells, i.e. hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). With the aim to exploit niche cell properties for experimental and potential clinical applications, we analyzed the potential of primary ECs alone and in combination with MSCs to support the ex vivo expansion/maintenance of human hematopoietic stem and progenitor cells (HSPCs). Even though a massive expansion of total CD34 HSPCs was observed, none of the tested culture conditions supported the expansion or maintenance of multipotent HSPCs. Instead, mainly lympho-myeloid primed progenitors (LMPPs) were expanded. Similarly, following transplantation into immunocompromised mice the percentage of multipotent HSPCs within the engrafted HSPC population was significantly decreased compared to the original graft. Consistent with the in vitro findings, a bias towards lympho-myeloid lineage potentials was observed. In our conditions, neither classical co-cultures of HSPCs with primary ECs or MSCs, even in combination, nor the xenograft environment in immunocompromised mice efficiently support the expansion of multipotent HSPCs. Instead, enhanced expansion and a consistent bias towards lympho-myeloid committed LMPPs were observed.
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http://dx.doi.org/10.1038/s41598-019-49221-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733927PMC
September 2019

Allogeneic transplantation of peripheral blood stem cell grafts results in a massive decrease of primitive hematopoietic progenitor frequencies in reconstituted bone marrows.

Bone Marrow Transplant 2020 01 21;55(1):100-109. Epub 2019 Aug 21.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

The success of allogeneic hematopoietic stem cell transplantation (alloSCT) is indicated by the reconstitution of the peripheral blood system of patients after alloSCT and the engraftment of hematopoietic stem and progenitor cells (HSPCs) into their bone marrow (BM). The number of CD34 cells is commonly used as surrogate for the content of hematopoietic stem cells in the grafts. During the last decade, several antigens (including CD133, CD45RA, CD38, and CD10) were identified allowing discrimination of different HSPC subpopulations within the human CD34 cell compartment. Although such studies increased our understanding of early human hematopoiesis tremendously, hardly any study dissected the CD34 compartment in the alloSCT setting. Consequently, we comprehensively analyzed the CD34 compartment in G-CSF-stimulated peripheral blood stem cell grafts of allogeneic donors, in BM samples of the respective recipients 4 weeks after alloSCT, and in BM samples of healthy donors. We demonstrate that alloSCT is associated with a dramatic shift from primitive to more mature HSPC types. Upon investigating whether the composition of engrafted CD34 cells has any impact on the incidence and severity of graft-versus-host disease, we did not find any correlation. However, more detailed analyses of the CD34 compartment may elucidate associations with other transplantation-related complications.
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http://dx.doi.org/10.1038/s41409-019-0645-7DOI Listing
January 2020

Distinct Spatio-Temporal Dynamics of Tumor-Associated Neutrophils in Small Tumor Lesions.

Front Immunol 2019 25;10:1419. Epub 2019 Jun 25.

Department of Otorhinolaryngology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Across a majority of cancer types tumor-associated neutrophils (TAN) are linked with poor prognosis. However, the underlying mechanisms, especially the intratumoral behavior of TAN, are largely unknown. Using intravital multiphoton imaging on a mouse model with neutrophil-specific fluorescence, we measured the migration of TAN in distinct compartments of solid tumor cell lesions . By longitudinally quantifying the infiltration and persistence of TAN into growing tumors in the same animals, we observed cells that either populated the peripheral stromal zone of the tumor (peritumoral TAN) or infiltrated into the tumor core (intratumoral TAN). Intratumoral TAN showed prolonged tumor-associated persistence and reduced motility compared to peritumoral TAN, whose velocity increased with tumor progression. Selective pharmacological blockade of CXCR2 receptors using AZD5069 profoundly inhibited recruitment of TAN into peritumoral regions, while intratumoral infiltration was only transiently attenuated and rebounded at later time points. Our findings unravel distinct spatial dynamics of TAN that are partially and differentially regulated via the CXCR2 signaling pathway.
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http://dx.doi.org/10.3389/fimmu.2019.01419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603174PMC
October 2020

Identification of the right cell sources for the production of therapeutically active extracellular vesicles in ischemic stroke.

Ann Transl Med 2019 May;7(9):188

Department of Neurology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

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http://dx.doi.org/10.21037/atm.2019.03.49DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545303PMC
May 2019

Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.

J Extracell Vesicles 2019 29;8(1):1609206. Epub 2019 Apr 29.

Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore.

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of and functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.
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http://dx.doi.org/10.1080/20013078.2019.1609206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493293PMC
April 2019

Individual Immune-Modulatory Capabilities of MSC-Derived Extracellular Vesicle (EV) Preparations and Recipient-Dependent Responsiveness.

Int J Mol Sci 2019 Apr 2;20(7). Epub 2019 Apr 2.

Institute for Transfusion Medicine, University Hospital Essen, 45147 Essen, Germany.

Treatment with extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been suggested as novel therapeutic option in acute inflammation-associated disorders due to their immune-modulatory capacities. As we have previously observed differences in the cytokine profile of independent MSC-EV preparations, functional differences of MSC-EV preparations have to be considered. To evaluate the immune-modulatory capabilities of specific MSC-EV preparations, reliable assays are required to characterize the functionality of MSC-EV preparations prior to administration to a patient. To this end, we established an in vitro assay evaluating the immune-modulatory capacities of MSC-EV preparations. Here, we compared the efficacy of four independent MSC-EV preparations to modulate the induction of T cell differentiation and cytokine production after phorbol 12-myristate 13-acetate (PMA)/Ionomycin stimulation of peripheral blood mononuclear cells (PBMC) derived from six healthy donors. Flow cytometric analyses revealed that the four MSC-EV preparations differentially modulate the expression of surface markers, such as CD45RA, on CD4+ and CD8+ T cells, resulting in shifts in the frequencies of effector and effector memory T cells. Moreover, cytokine profile in T cell subsets was affected in a MSC-EV-specific manner exclusively in CD8+ naïve T cells. Strikingly, hierarchical clustering revealed that the T cell response towards the MSC-EV preparations largely varied among the different PBMC donors. Thus, besides defining functional activity of MSC-EV preparations, it will be crucial to test whether patients intended for treatment with MSC-EV preparations are in principal competent to respond to the envisioned MSC-EV therapy.
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http://dx.doi.org/10.3390/ijms20071642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479947PMC
April 2019

Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours.

J Extracell Vesicles 2019 21;8(1):1588555. Epub 2019 Mar 21.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Cells release heterogeneous nano-sized vesicles either as exosomes, being derived from endosomal compartments, or through budding from the plasma membrane as so-called microvesicles, commonly referred to as extracellular vesicles (EVs). EVs are known for their important roles in mammalian physiology and disease pathogenesis and provide a potential biomarker source in cancer patients. EVs are generally often analysed in bulk using Western blotting or by bead-based flow-cytometry or, with limited parameters, through nanoparticle tracking analysis. Due to their small size, single EV analysis is technically highly challenging. Here we demonstrate imaging flow cytometry (IFCM) to be a robust, multiparametric technique that allows analysis of single EVs and the discrimination of distinct EV subpopulations. We used IFCM to analyse the tetraspanin (CD9, CD63, CD81) surface profiles on EVs from human and murine cell cultures as well as plasma samples. The presence of EV subpopulations with specific tetraspanin profiles suggests that EV-mediated cellular responses are tightly regulated and dependent on cell environment. We further demonstrate that EVs with double positive tetraspanin expression (CD63/CD81) are enriched in cancer cell lines and patient plasma samples. In addition, we used IFCM to detect tumour-specific GFP-labelled EVs in the blood of mice bearing syngeneic intracerebral gliomas, indicating that this technique allows unprecedented disease modelling. In summary, our study highlights the heterogeneous and adaptable nature of EVs according to their marker profile and demonstrates that IFCM facilitates multiparametric phenotyping of EVs not only but also in patient plasma at a single EV level, with the potential for future functional studies and clinically relevant applications. EDTA = ethylenediamine tetraacetic acid.
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http://dx.doi.org/10.1080/20013078.2019.1588555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442086PMC
March 2019

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material.

J Extracell Vesicles 2019 21;8(1):1587567. Epub 2019 Mar 21.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.
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http://dx.doi.org/10.1080/20013078.2019.1587567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442110PMC
March 2019

Annexin A1 as Neuroprotective Determinant for Blood-Brain Barrier Integrity in Neonatal Hypoxic-Ischemic Encephalopathy.

J Clin Med 2019 Jan 24;8(2). Epub 2019 Jan 24.

Department of Pediatrics, Maastricht University Medical Center, 6202 AZ Maastricht, The Netherlands.

Blood-brain barrier (BBB) disruption is associated with hypoxia-ischemia (HI) induced brain injury and life-long neurological pathologies. Treatment options are limited. Recently, we found that mesenchymal stem/stromal cell derived extracellular vesicles (MSC-EVs) protected the brain in ovine fetuses exposed to HI. We hypothesized that Annexin A1 (ANXA1), present in MSC-EVs, contributed to their therapeutic potential by targeting the ANXA1/Formyl peptide receptor (FPR), thereby preventing loss of the BBB integrity. Cerebral ANXA1 expression and leakage of albumin into the fetal ovine brain parenchyma after HI were analyzed by immunohistochemistry. For mechanistic insights, barrier integrity of primary fetal endothelial cells was assessed after oxygen-glucose deprivation (OGD) followed by treatment with MSC-EVs or human recombinant ANXA1 in the presence or absence of FPR inhibitors. Our study revealed that BBB integrity was compromised after HI which was improved by MSC-EVs containing ANXA1. Treatment with these MSC-EVs or ANXA1 improved BBB integrity after OGD, an effect abolished by FPR inhibitors. Furthermore, endogenous ANXA1 was depleted within 24 h after induction of HI in cerebovasculature and ependyma and upregulated 72 h after HI in microglia. Targeting ANXA1/FPR with ANXA1 in the immature brain has great potential in preventing BBB loss and concomitant brain injury following HI.
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http://dx.doi.org/10.3390/jcm8020137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406389PMC
January 2019

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

Authors:
Clotilde Théry Kenneth W Witwer Elena Aikawa Maria Jose Alcaraz Johnathon D Anderson Ramaroson Andriantsitohaina Anna Antoniou Tanina Arab Fabienne Archer Georgia K Atkin-Smith D Craig Ayre Jean-Marie Bach Daniel Bachurski Hossein Baharvand Leonora Balaj Shawn Baldacchino Natalie N Bauer Amy A Baxter Mary Bebawy Carla Beckham Apolonija Bedina Zavec Abderrahim Benmoussa Anna C Berardi Paolo Bergese Ewa Bielska Cherie Blenkiron Sylwia Bobis-Wozowicz Eric Boilard Wilfrid Boireau Antonella Bongiovanni Francesc E Borràs Steffi Bosch Chantal M Boulanger Xandra Breakefield Andrew M Breglio Meadhbh Á Brennan David R Brigstock Alain Brisson Marike Ld Broekman Jacqueline F Bromberg Paulina Bryl-Górecka Shilpa Buch Amy H Buck Dylan Burger Sara Busatto Dominik Buschmann Benedetta Bussolati Edit I Buzás James Bryan Byrd Giovanni Camussi David Rf Carter Sarah Caruso Lawrence W Chamley Yu-Ting Chang Chihchen Chen Shuai Chen Lesley Cheng Andrew R Chin Aled Clayton Stefano P Clerici Alex Cocks Emanuele Cocucci Robert J Coffey Anabela Cordeiro-da-Silva Yvonne Couch Frank Aw Coumans Beth Coyle Rossella Crescitelli Miria Ferreira Criado Crislyn D'Souza-Schorey Saumya Das Amrita Datta Chaudhuri Paola de Candia Eliezer F De Santana Olivier De Wever Hernando A Del Portillo Tanguy Demaret Sarah Deville Andrew Devitt Bert Dhondt Dolores Di Vizio Lothar C Dieterich Vincenza Dolo Ana Paula Dominguez Rubio Massimo Dominici Mauricio R Dourado Tom Ap Driedonks Filipe V Duarte Heather M Duncan Ramon M Eichenberger Karin Ekström Samir El Andaloussi Celine Elie-Caille Uta Erdbrügger Juan M Falcón-Pérez Farah Fatima Jason E Fish Miguel Flores-Bellver András Försönits Annie Frelet-Barrand Fabia Fricke Gregor Fuhrmann Susanne Gabrielsson Ana Gámez-Valero Chris Gardiner Kathrin Gärtner Raphael Gaudin Yong Song Gho Bernd Giebel Caroline Gilbert Mario Gimona Ilaria Giusti Deborah Ci Goberdhan André Görgens Sharon M Gorski David W Greening Julia Christina Gross Alice Gualerzi Gopal N Gupta Dakota Gustafson Aase Handberg Reka A Haraszti Paul Harrison Hargita Hegyesi An Hendrix Andrew F Hill Fred H Hochberg Karl F Hoffmann Beth Holder Harry Holthofer Baharak Hosseinkhani Guoku Hu Yiyao Huang Veronica Huber Stuart Hunt Ahmed Gamal-Eldin Ibrahim Tsuneya Ikezu Jameel M Inal Mustafa Isin Alena Ivanova Hannah K Jackson Soren Jacobsen Steven M Jay Muthuvel Jayachandran Guido Jenster Lanzhou Jiang Suzanne M Johnson Jennifer C Jones Ambrose Jong Tijana Jovanovic-Talisman Stephanie Jung Raghu Kalluri Shin-Ichi Kano Sukhbir Kaur Yumi Kawamura Evan T Keller Delaram Khamari Elena Khomyakova Anastasia Khvorova Peter Kierulf Kwang Pyo Kim Thomas Kislinger Mikael Klingeborn David J Klinke Miroslaw Kornek Maja M Kosanović Árpád Ferenc Kovács Eva-Maria Krämer-Albers Susanne Krasemann Mirja Krause Igor V Kurochkin Gina D Kusuma Sören Kuypers Saara Laitinen Scott M Langevin Lucia R Languino Joanne Lannigan Cecilia Lässer Louise C Laurent Gregory Lavieu Elisa Lázaro-Ibáñez Soazig Le Lay Myung-Shin Lee Yi Xin Fiona Lee Debora S Lemos Metka Lenassi Aleksandra Leszczynska Isaac Ts Li Ke Liao Sten F Libregts Erzsebet Ligeti Rebecca Lim Sai Kiang Lim Aija Linē Karen Linnemannstöns Alicia Llorente Catherine A Lombard Magdalena J Lorenowicz Ákos M Lörincz Jan Lötvall Jason Lovett Michelle C Lowry Xavier Loyer Quan Lu Barbara Lukomska Taral R Lunavat Sybren Ln Maas Harmeet Malhi Antonio Marcilla Jacopo Mariani Javier Mariscal Elena S Martens-Uzunova Lorena Martin-Jaular M Carmen Martinez Vilma Regina Martins Mathilde Mathieu Suresh Mathivanan Marco Maugeri Lynda K McGinnis Mark J McVey David G Meckes Katie L Meehan Inge Mertens Valentina R Minciacchi Andreas Möller Malene Møller Jørgensen Aizea Morales-Kastresana Jess Morhayim François Mullier Maurizio Muraca Luca Musante Veronika Mussack Dillon C Muth Kathryn H Myburgh Tanbir Najrana Muhammad Nawaz Irina Nazarenko Peter Nejsum Christian Neri Tommaso Neri Rienk Nieuwland Leonardo Nimrichter John P Nolan Esther Nm Nolte-'t Hoen Nicole Noren Hooten Lorraine O'Driscoll Tina O'Grady Ana O'Loghlen Takahiro Ochiya Martin Olivier Alberto Ortiz Luis A Ortiz Xabier Osteikoetxea Ole Østergaard Matias Ostrowski Jaesung Park D Michiel Pegtel Hector Peinado Francesca Perut Michael W Pfaffl Donald G Phinney Bartijn Ch Pieters Ryan C Pink David S Pisetsky Elke Pogge von Strandmann Iva Polakovicova Ivan Kh Poon Bonita H Powell Ilaria Prada Lynn Pulliam Peter Quesenberry Annalisa Radeghieri Robert L Raffai Stefania Raimondo Janusz Rak Marcel I Ramirez Graça Raposo Morsi S Rayyan Neta Regev-Rudzki Franz L Ricklefs Paul D Robbins David D Roberts Silvia C Rodrigues Eva Rohde Sophie Rome Kasper Ma Rouschop Aurelia Rughetti Ashley E Russell Paula Saá Susmita Sahoo Edison Salas-Huenuleo Catherine Sánchez Julie A Saugstad Meike J Saul Raymond M Schiffelers Raphael Schneider Tine Hiorth Schøyen Aaron Scott Eriomina Shahaj Shivani Sharma Olga Shatnyeva Faezeh Shekari Ganesh Vilas Shelke Ashok K Shetty Kiyotaka Shiba Pia R-M Siljander Andreia M Silva Agata Skowronek Orman L Snyder Rodrigo Pedro Soares Barbara W Sódar Carolina Soekmadji Javier Sotillo Philip D Stahl Willem Stoorvogel Shannon L Stott Erwin F Strasser Simon Swift Hidetoshi Tahara Muneesh Tewari Kate Timms Swasti Tiwari Rochelle Tixeira Mercedes Tkach Wei Seong Toh Richard Tomasini Ana Claudia Torrecilhas Juan Pablo Tosar Vasilis Toxavidis Lorena Urbanelli Pieter Vader Bas Wm van Balkom Susanne G van der Grein Jan Van Deun Martijn Jc van Herwijnen Kendall Van Keuren-Jensen Guillaume van Niel Martin E van Royen Andre J van Wijnen M Helena Vasconcelos Ivan J Vechetti Tiago D Veit Laura J Vella Émilie Velot Frederik J Verweij Beate Vestad Jose L Viñas Tamás Visnovitz Krisztina V Vukman Jessica Wahlgren Dionysios C Watson Marca Hm Wauben Alissa Weaver Jason P Webber Viktoria Weber Ann M Wehman Daniel J Weiss Joshua A Welsh Sebastian Wendt Asa M Wheelock Zoltán Wiener Leonie Witte Joy Wolfram Angeliki Xagorari Patricia Xander Jing Xu Xiaomei Yan María Yáñez-Mó Hang Yin Yuana Yuana Valentina Zappulli Jana Zarubova Vytautas Žėkas Jian-Ye Zhang Zezhou Zhao Lei Zheng Alexander R Zheutlin Antje M Zickler Pascale Zimmermann Angela M Zivkovic Davide Zocco Ewa K Zuba-Surma

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Kraków, Poland.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Induction of herpes simplex virus type 1 cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine.

Nanomedicine 2019 02 27;16:138-148. Epub 2018 Dec 27.

Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; Department of Infectious Diseases, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany. Electronic address:

Herpes simplex viruses 1 and 2 are among the most ubiquitous human infections and persist lifelong in their host. Upon primary infection or reactivation from ganglia, the viruses spread by direct cell-cell contacts (cell-to-cell spread) and thus escape from the host immune response. We have developed a monoclonal antibody (mAb 2c), which inhibits the HSV cell-to-cell spread, thereby protecting from lethal genital infection and blindness in animal models. In the present study we have designed a nanoparticle-based vaccine to induce protective antibody responses exceeding the cell-to-cell spread inhibiting properties of mAb 2c. We used biodegradable calcium phosphate (CaP) nanoparticles coated with a synthetic peptide that represents the conformational epitope on HSV-1 gB recognized by mAb 2c. The CaP nanoparticles additionally contained a TLR-ligand CpG and were formulated with adjuvants to facilitate the humoral immune response. This vaccine effectively protected mice from lethal HSV-1 infection by inducing cell-to-cell spread inhibiting antibodies.
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http://dx.doi.org/10.1016/j.nano.2018.12.002DOI Listing
February 2019

Remote ischaemic preconditioning increases serum extracellular vesicle concentrations with altered micro-RNA signature in CABG patients.

Acta Anaesthesiol Scand 2019 04 11;63(4):483-492. Epub 2018 Dec 11.

Klinik für Anästhesiologie und Intensivmedizin, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany.

Background: Remote ischaemic preconditioning (RIPC) can attenuate myocardial ischaemia/reperfusion injury but its underlying mechanisms remain largely unknown. Recently, extracellular vesicles (EVs) containing microRNAs (miRNAs) were shown to mediate distant intercellular communication that may be involved in cardioprotection. We tested the hypothesis that RIPC in anaesthetized patients undergoing coronary artery bypass (CABG) surgery results in the release of EVs from the ischaemic/reperfused arm into the blood stream harbouring cardioprotective miRNAs.

Methods: In 58 patients randomised to RIPC (three 5/5 minutes episodes of left arm ischaemia/reperfusion by suprasystolic blood pressure cuff inflations/deflations) or Sham, a subprotocol comprising of parallel right radial artery and regional (left subclavian) venous blood sampling before (awake) and 5 and 60 minutes after RIPC/Sham during isoflurane/sufentanil anaesthesia could be completed. EVs were extracted by polymer-based precipitation methods, their concentrations measured, and their miRNA signature analysed.

Results: Five minutes after RIPC, regional venous EV concentrations downstream from the cuff increased and arterial concentrations increased after 60 minutes (fold change [fc]: RIPC: 1.33 ± 0.5, Sham: 0.91 ± 0.31; P = 0.003 for interaction). Already 5 minutes after RIPC, expression of 26 miRNAs (threshold fc: 3.0, P < 0.05) isolated from EVs including the cardioprotective miR-21 had increased. RIPC also decreased postoperative Troponin I concentrations (AUC RIPC: 336 ng/mL × 72 hours ± 306 vs Sham: 713 ± 1013; P  =  0.041).

Conclusions: Remote ischaemic preconditioning increases serum EV concentrations, most likely by early EV release from the patients' left (RIPC) arm, alters their miRNA signature, and is associated with myocardial protection. Thus, an increased EV concentration with an altered miR-signature may mediate the RIPC effect.
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http://dx.doi.org/10.1111/aas.13296DOI Listing
April 2019