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1.
Proc Natl Acad Sci U S A ; 120(40): e2215421120, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37756334

RESUMO

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , Interleucina-27 , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Histonas , Plaquetas , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética
2.
J Immunol ; 206(12): 2839-2851, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34117106

RESUMO

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.


Assuntos
Linfócitos B/imunologia , Sangue Fetal/imunologia , Imunoglobulinas/imunologia , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos B/imunologia
3.
Stroke ; 53(1): e14-e18, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34847707

RESUMO

BACKGROUND AND PURPOSE: Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce ischemic neuroprotection in mice by modulating the brain infiltration of leukocytes and, specifically polymorphonuclear neutrophils. So far, effects of MSC-sEVs were only studied in young ischemic rodents. We herein examined the effects of MSC-sEVs in aged mice. METHODS: Male and female C57Bl6/j mice (8-10 weeks or 15-24 months) were exposed to transient intraluminal middle cerebral artery occlusion. Vehicle or sEVs (equivalent of 2×106 MSCs) were intravenously administered. Neurological deficits, ischemic injury, blood-brain barrier integrity, brain leukocyte infiltration, and blood leukocyte responses were evaluated over up to 7 days. RESULTS: MSC-sEV delivery reduced neurological deficits, infarct volume, brain edema, and neuronal injury in young and aged mice of both sexes, when delivered immediately postreperfusion or with 6 hours delay. MSC-sEVs decreased leukocyte and specifically polymorphonuclear neutrophil, monocyte, and macrophage infiltrates in ischemic brains of aged mice. In peripheral blood, the number of monocytes and activated T cells was significantly reduced by MSC-sEVs. CONCLUSIONS: MSC-sEVs induce postischemic neuroprotection and anti-inflammation in aged mice.


Assuntos
Envelhecimento/fisiologia , Vesículas Extracelulares/metabolismo , Infarto da Artéria Cerebral Média/terapia , Células-Tronco Mesenquimais/citologia , Neuroproteção/fisiologia , Animais , Encéfalo/irrigação sanguínea , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neurônios/citologia
4.
Stroke ; 51(6): 1825-1834, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32312217

RESUMO

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% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) 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.


Assuntos
Barreira Hematoencefálica , Isquemia Encefálica , Vesículas Extracelulares , Células-Tronco Mesenquimais/metabolismo , Neuroproteção , Neutrófilos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Isquemia Encefálica/sangue , Isquemia Encefálica/patologia , Isquemia Encefálica/terapia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Vesículas Extracelulares/transplante , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Neutrófilos/patologia
5.
Acta Anaesthesiol Scand ; 63(4): 483-492, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30548252

RESUMO

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  = â€Š0.041). 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.


Assuntos
Ponte de Artéria Coronária , Vesículas Extracelulares , Precondicionamento Isquêmico Miocárdico/métodos , MicroRNAs/sangue , Idoso , Idoso de 80 Anos ou mais , Anestesia Geral , Anestésicos Inalatórios , Anestésicos Intravenosos , Método Duplo-Cego , Feminino , Traumatismos Cardíacos/sangue , Humanos , Isoflurano , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/sangue , Sufentanil , Troponina I/sangue
6.
Mol Ther ; 24(8): 1423-34, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27112062

RESUMO

Recent clinical trials with chimeric antigen receptor (CAR) redirected T cells targeting CD19 revealed particular efficacy in the treatment of leukemia/lymphoma, however, were accompanied by a lasting depletion of healthy B cells. We here explored CD30 as an alternative target, which is validated in lymphoma therapy and expressed by a broad variety of Hodgkin's and non-Hodgkin's lymphomas. As a safty concern, however, CD30 is also expressed by lymphocytes and hematopoietic stem and progenitor cells (HSPCs) during activation. We revealed that HRS3scFv-derived CAR T cells are superior since they were not blocked by soluble CD30 and did not attack CD30(+) HSPCs while eliminating CD30(+) lymphoma cells. Consequently, normal hemato- and lymphopoiesis was not affected in the long-term in the humanized mouse; the number of blood B and T cells remained unchanged. We provide evidence that the CD30(+) HSPCs are protected against a CAR T-cell attack by substantially lower CD30 levels than lymphoma cells and higher levels of the granzyme B inactivating SP6/PI9 serine protease, which furthermore increased upon activation. Taken together, adoptive cell therapy with anti-CD30 CAR T cells displays a superior therapeutic index in the treatment of CD30(+) malignancies leaving healthy activated lymphocytes and HSPCs unaffected.

7.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33538775

RESUMO

Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging.


Assuntos
Envelhecimento/imunologia , Linfócitos B/imunologia , Memória Imunológica , Baço/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Doadores de Sangue , Linhagem Celular , Criança , Pré-Escolar , Técnicas de Cocultura , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pessoa de Meia-Idade , Fenótipo , Receptores de Complemento 3d/metabolismo , Baço/patologia , Adulto Jovem
8.
PLoS One ; 15(2): e0228674, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040489

RESUMO

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.


Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Oligodesoxirribonucleotídeos/farmacologia , Animais , Células da Medula Óssea/citologia , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Receptores CXCR4/metabolismo , Receptor Toll-Like 9/metabolismo
9.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211891

RESUMO

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.


Assuntos
Centríolos/enzimologia , Cílios/enzimologia , Células Epiteliais/enzimologia , Mitose , Quinases Relacionadas a NIMA/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Animais , Sítios de Ligação , Linhagem Celular , Centríolos/genética , Cílios/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Células-Tronco Hematopoéticas/enzimologia , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/enzimologia , Camundongos , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/genética , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais , Fatores de Tempo
10.
Acta Biomater ; 111: 349-362, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32413579

RESUMO

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-NH2. 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.


Assuntos
Ouro , Nanopartículas Metálicas , Barreira Hematoencefálica , Encéfalo , Núcleo Celular , Células Endoteliais , Humanos , Esferoides Celulares
11.
PLoS One ; 15(2): e0228948, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32059016

RESUMO

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.


Assuntos
Vesículas Extracelulares/fisiologia , Isquemia/metabolismo , Miócitos Cardíacos/fisiologia , Idoso , Anestesia/métodos , Animais , Apoptose/fisiologia , Ponte de Artéria Coronária/métodos , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Hipóxia/metabolismo , Precondicionamento Isquêmico/métodos , Isoflurano/farmacologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Propofol/farmacologia , Ratos
12.
J Extracell Vesicles ; 8(1): 1587567, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30949308

RESUMO

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.

13.
Front Immunol ; 9: 1326, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29951064

RESUMO

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

14.
Front Oncol ; 5: 234, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26539411

RESUMO

Widespread metastasis is the leading course of death in many types of cancer, including malignant melanoma. The process of metastasis can be divided into a number of complex cell biological events, collectively termed the "invasion-metastasis cascade." Previous reports have characterized the capability of anchorage-independent growth of cancer cells in vitro as a key characteristic of highly aggressive tumor cells, particularly with respect to metastatic potential. Biological heterogeneity as well as drastic alterations in cell adhesion of disseminated cancer cells support escape mechanisms for metastases to overcome conventional therapies. Here, we show that exclusively the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) splice variant CEACAM1-4L supports an anchorage-independent signature in malignant melanoma. These results highlight important variant-specific modulatory functions of CEACAM1 for metastatic spread in patients suffering malignant melanoma.

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