scholarly journals CSIG-11. CENTRAL NERVOUS SYSTEM TUMOR PATIENTS HAVE ELEVATED LEVELS OF CIRCULATING EXTRACELLULAR VESICLES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi46-vi46
Author(s):  
Franz Ricklefs ◽  
Cecile Maire ◽  
Katharina Kolbe ◽  
Mareike Holz ◽  
Rudolph Reimer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Molecular Subtype ◽  
Cell Types ◽  
Central Nervous System Tumor ◽  
Healthy Controls ◽  
Double Positive ◽  
Tumor Patients ◽  
System Tumor

Abstract BACKGROUND We previously demonstrated that extracellular vesicles (EV) from CNS tumors reflect the molecular subtype of the original tumor and mediate an exchange of pro-oncogenic signals. EVs are commonly characterized by nanoparticle analysis (NTA), electron microscopy and tetraspanin markers, including CD9, CD81 and CD63. It is unclear, however, to what extent circulating tumor EVs are utilizable for diagnostic purposes and how their marker profile overlaps with EVs derived from other cell types. Aiming to define markers that allow distinction and enrichment of glioma EVs from patient blood, we utilized Imaging Flow Cytometry (IFC) to discriminate single EVs via multiple surface markers. METHODS EVs were isolated from blood of patients suffering from glioblastoma (n=24), anaplastic astrocytoma (n=8), brain metastasis (n=7), meningioma (n=12), pituitary gland tumor (n=11), epilepsy (n=11) and from healthy controls (n=18) and were analyzed by IFC, immunoblotting, electron microscopy and NTA. In addition, circulating EVs from PALM-GFP-GL261 and PALM-GFP-CT2A tumor-bearing mice (n=5) as well as from glioblastoma stem-like (GSC) cultures (n=4), neural stem cells (NSC), cerebral endothelial cells (cEC) and T-cells (n=4) were characterized. RESULTS CNS tumor patients have significantly elevated levels of circulating EVs (P < 0.001), as measured by NTA and IFC. In particular, the proportion of double positive CD9+/CD81+, CD9+/CD63+and CD63+/CD81+EVs is increased in glioblastoma patients (p=0.018) compared with healthy controls[L1]. In accordance, cultured GSCs secrete increased levels of CD9+/CD81+EVs in vitro. In the two syngeneic murine PALM-GFP glioma models, only 0.1-0.01% of circulating plasma EVs were found to be derived from intracranial tumors, underlining the need to identify markers that can enrich tumor-specific EVs for molecular profiling. CONCLUSION Glioma patients display increased levels of circulating plasma EVs that can be profiled by IFC, which is a unique and novel technique that facilitates discrimination of different EV subpopulations.

scholarly journals BIOM-09. MULTIPLEX ANALYSIS OF CSF EXTRACELLULAR VESICLES OF INTRASPINAL TUMORS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii3-ii3
Author(s):  
Franz Ricklefs ◽  
Ines Stevic ◽  
Christian Mende ◽  
Joshua Welsh ◽  
Jennifer Jones ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Molecular Subtype ◽  
Cell Communication ◽  
Normal Pressure ◽  
Tumor Patients ◽  
Intraspinal Tumor ◽  
Hla Dr ◽  
Multiplex Bead

Abstract BACKGROUND: Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. We previously demonstrated that extracellular vesicles (EV) from central nervous system tumors reflect the molecular subtype of the original tumor and mediate an exchange of pro-oncogenic signals. Their implication as biomarkers in tumor disease is under current investigation. It is unclear, however, to what extent cerebrospinal fluid (CSF) EVs from intraspinal tumors are utilizable for diagnostical purposes and how their marker profiles overlap with EVs derived from non tumorous EVs. We analyzed CSF EVs of intraspinal tumors to define CSF EV profiles that allow tumor subtype classification. METHODS: EVs were isolated from CSF of patients suffering from intraspinal meningioma (n=5), ependymoma (n=7) and neurinoma (n=5). Patients suffering from normal pressure hydrocephalus were used as controls (n=5). EVs were analyzed by multiplex bead based assay, immunoblotting, electron microscopy and NTA. RESULTS: CSF EVs were 97.21 ± 3.37nm (intraspinal tumor patients) and 101.6 ± 3.68nm (controls) in sizes and showed vesicular structures by electron microscopy. Particle number were not significantly different between both groups (p = 0.103). Using our 37 protein mutliplex EV profiling kit we found 29 proteins to be expressed in a sufficient manner on CSF EVs. CSF EVs of intraspinal meningioma showed elevated CD62P, HLA-DR, CD40, CD42a and CD45 expression levels, while ependymoma showed decreased levels of CD9, CD63, CD81, whereas neurinomas had elevated levels of SSEA-3 and CD25. CONCLUSION: This is the first comprehensive analysis of CSF EV of intraspinal tumor patients. CSF EV display distinct subpopulations that may allow tumor classification and long-term surveillance. However as tumor-specific EVs may be rare, there is still the need to identify markers that can enrich tumor-specific EVs for molecular profiling.

scholarly journals Synthesis and Evaluation of AlgNa-g-poly(QCL-co-HEMA) Hydrogels as Platform for Chondrocyte Proliferation and Controlled Release of Betamethasone

2021 ◽  
Vol 22 (11) ◽  
pp. 5730
Author(s):  
Jomarien García-Couce ◽  
Marioly Vernhes ◽  
Nancy Bada ◽  
Lissette Agüero ◽  
Oscar Valdés ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Controlled Release ◽  
Biomedical Applications ◽  
Release Kinetics ◽  
Cell Types ◽  
Tissue Engineering Scaffolds ◽  
Almost All ◽  
Scanning Electron

Hydrogels obtained from combining different polymers are an interesting strategy for developing controlled release system platforms and tissue engineering scaffolds. In this study, the applicability of sodium alginate-g-(QCL-co-HEMA) hydrogels for these biomedical applications was evaluated. Hydrogels were synthesized by free-radical polymerization using a different concentration of the components. The hydrogels were characterized by Fourier transform-infrared spectroscopy, scanning electron microscopy, and a swelling degree. Betamethasone release as well as the in vitro cytocompatibility with chondrocytes and fibroblast cells were also evaluated. Scanning electron microscopy confirmed the porous surface morphology of the hydrogels in all cases. The swelling percent was determined at a different pH and was observed to be pH-sensitive. The controlled release behavior of betamethasone from the matrices was investigated in PBS media (pH = 7.4) and the drug was released in a controlled manner for up to 8 h. Human chondrocytes and fibroblasts were cultured on the hydrogels. The MTS assay showed that almost all hydrogels are cytocompatibles and an increase of proliferation in both cell types after one week of incubation was observed by the Live/Dead® assay. These results demonstrate that these hydrogels are attractive materials for pharmaceutical and biomedical applications due to their characteristics, their release kinetics, and biocompatibility.

scholarly journals Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

2019 ◽  
Author(s):  
Benedikt Kirchner ◽  
Dominik Buschmann ◽  
Vijay Paul ◽  
Michael W. Pfaffl
Keyword(s):  
Extracellular Vesicles ◽  
Expression Profiles ◽  
Bovine Milk ◽  
Cell Types ◽  
Specific Protein ◽  
In Vivo Experiments ◽  
Neonatal Calves ◽  
Almost All

Abstract Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

Hypercoaulability state in central nervous system tumor patients

1997 ◽  
Vol 99 ◽  
pp. S251
Author(s):  
M.E. Kusak ◽  
J.M. Alonso ◽  
D. Santamarta ◽  
I. Recio ◽  
J.M. Borrás ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Central Nervous System Tumor ◽  
Tumor Patients ◽  
System Tumor

scholarly journals Calciprotein particle inhibition explains magnesium-mediated protection against vascular calcification

2019 ◽  
Vol 35 (5) ◽  
pp. 765-773 ◽  
Author(s):  
Anique D ter Braake ◽  
Coby Eelderink ◽  
Lara W Zeper ◽  
Andreas Pasch ◽  
Stephan J L Bakker ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Chronic Kidney Disease ◽  
Smooth Muscle ◽  
Cardiovascular Risk ◽  
Kidney Disease ◽  
Vascular Calcification ◽  
Healthy Controls ◽  
Gene And Protein Expression ◽  
Calciprotein Particles

Abstract Background Phosphate (Pi) toxicity is a strong determinant of vascular calcification development in chronic kidney disease (CKD). Magnesium (Mg2+) may improve cardiovascular risk via vascular calcification. The mechanism by which Mg2+ counteracts vascular calcification remains incompletely described. Here we investigated the effects of Mg2+ on Pi and secondary crystalline calciprotein particles (CPP2)-induced calcification and crystal maturation. Methods Vascular smooth muscle cells (VSMCs) were treated with high Pi or CPP2 and supplemented with Mg2+ to study cellular calcification. The effect of Mg2+ on CPP maturation, morphology and composition was studied by medium absorbance, electron microscopy and energy dispersive spectroscopy. To translate our findings to CKD patients, the effects of Mg2+ on calcification propensity (T50) were measured in sera from CKD patients and healthy controls. Results Mg2+ supplementation prevented Pi-induced calcification in VSMCs. Mg2+ dose-dependently delayed the maturation of primary CPP1 to CPP2 in vitro. Mg2+ did not prevent calcification and associated gene and protein expression when added to already formed CPP2. Confirmatory experiments in human serum demonstrated that the addition of 0.2 mmol/L Mg2+ increased T50 from healthy controls by 51 ± 15 min (P &lt; 0.05) and CKD patients by 44 ± 13 min (P &lt; 0.05). Each further 0.2 mmol/L addition of Mg2+ led to further increases in both groups. Conclusions Our results demonstrate that crystalline CPP2 mediates Pi-induced calcification in VSMCs. In vitro, Mg2+ delays crystalline CPP2 formation and thereby prevents Pi-induced calcification.

scholarly journals The influence of jasmonic acid on the amount and the distribution of cysteine proteinase PLCP-2 in healthy and PVYNTN infected potato plants (Solanum tuberosum L.)

2002 ◽  
Vol 38 (SI 1 - 6th Conf EFPP 2002) ◽  
pp. S95-S98
Author(s):  
M. Pompe-Novak ◽  
M. Tušek-Žnidarič ◽  
B. Štrukelj ◽  
M. Ravnikar
Keyword(s):  
Electron Microscopy ◽  
Solanum Tuberosum ◽  
Jasmonic Acid ◽  
Cell Walls ◽  
Cysteine Proteinase ◽  
Solanum Tuberosum L ◽  
Cell Types ◽  
Protein Bodies ◽  
Potato Plants

The localization of cysteine proteinase PLCP-2 was investigated in potato plants (Solanum tuberosum L.) cultivar Désirée by electron microscopy. Healthy and PVY<sup>NTN</sup> infected potato plants were grown in vitro on media with or without a supplement of jasmonic acid. We had already shown that PLCP-2 is present in leaves, stems, tips of shoots and tips of roots of healthy and PVY<sup>NTN</sup> infected plants. It was detected in various cell types in protein bodies in vacuoles, in cytoplasm and in cell walls. There were significantly larger amounts of PLCP-2 in plants grown on medium with a supplement of jasmonic acid in both healthy and virus infected plants. More protein bodies in vacuoles were found in plants grown on medium with addition of jasmonic acid.

scholarly journals Human Heart Explant-Derived Extracellular Vesicles: Characterization and Effects on the In Vitro Recellularization of Decellularized Heart Valves

2019 ◽  
Vol 20 (6) ◽  
pp. 1279 ◽  
Author(s):  
Amanda Leitolis ◽  
Paula Suss ◽  
João Roderjan ◽  
Addeli Angulski ◽  
Francisco da Costa ◽  
...  
Keyword(s):  
Wound Healing ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Tissue Regeneration ◽  
Human Heart ◽  
Heart Valves ◽  
Enrichment Analysis ◽  
Cell Types

Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.

Surface topography of normal and neoplastic human anterior pituitary cells maintained in vitro

1978 ◽  
Vol 49 (2) ◽  
pp. 169-178 ◽  
Author(s):  
Robert D. Harris ◽  
Edward L. Seljeskog ◽  
Kenneth J. Murray ◽  
Shelley N. Chou ◽  
William P. Cunningham ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Surface Topography ◽  
Metastatic Cancer ◽  
Pituitary Tumors ◽  
Cell Types ◽  
Pituitary Cells ◽  
Content Type ◽  
Transmission Electron ◽  
Human Anterior Pituitary

✓ Pituitary tissues were obtained from 25 patients who underwent surgery for excision of pituitary macroadenomas, selective excision of microadenomas, or removal of a normal gland for palliation of metastatic cancer. Cells thus obtained were maintained in vitro for varying intervals, fixed, and examined by light (phase contrast), microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Previous SEM reports indicate that surface topography of in vitro neoplastic cells displays features that may correlate with neoplastic behavior. Cultured normal and pituitary tumor cells did not display these surface differences, with one exception, a prolactin-secreting microadenoma. Characteristic patterns for the cell populations were identified. Certain cell types appeared in all the cultures: 1) large and small granule-containing cells; 2) flat and irregular agranular cells; 3) spindle-shaped cells; and 4) spherical, irregularly surfaced cells. In one case of an endocrine-inactive juvenile pituitary chromophobe adenoma, unique cells were observed. Surface topography did not appear to be of predictive value in determining the neoplastic character of pituitary tumors.

scholarly journals Role of Prolactin and Growth Hormone on Thymus Physiology

1998 ◽  
Vol 6 (3-4) ◽  
pp. 317-323 ◽  
Author(s):  
Valéria De Mello-Coelho ◽  
Wilson Savino ◽  
Marie-Catherine Postel-Vinay ◽  
Mireille Dardenne
Keyword(s):  
Growth Hormone ◽  
Epithelial Cells ◽  
Cell Types ◽  
Pituitary Hormones ◽  
Thymic Epithelial Cells ◽  
Double Positive ◽  
Gh Treatment ◽  
Thymic Hormone

Intrathymic T-cell differentiation is under the control of the thymic microenvironment, which acts on maturing thymocytes via membrane as well as soluble products. Increasing data show that this process can be modulated by classical hormones, as exemplified herein by prolactin (PRL) and growth hormone (GH), largely secreted by the pituitary gland.Both PRL and GH stimulate the secretion of thymulin, a thymic hormone produced by thymic epithelial cells. Conversely, low levels of circulating thymulin parallel hypopituitary states. Interestingly, the enhancing effects of GH on thymulin seem to be mediated by insulinlike growth factor (IGF-1) since they can be abrogated with anti-IGF-1 or anti-IGF-l-receptor antibodies. The influence of PRL and GH on the thymic epithelium is pleiotropic: PRL enhancesin vivothe expression of high-molecular-weight cytokeratins and stimulatesin vitroTEC proliferation, an effect that is shared by GH and IGF-1.Differentiating T cells are also targets for the intrathymic action of PRL and GH.In vivoinoculation of a rat pituitary cell line into old rats results in restoration of the thymus, including differentiation of CD4-CD8-thymocytes into CD4+CD8+cells. Furthermore, PRL may regulate the maintenance of thymocyte viability during the double-positive stage of thymocyte differentiation.Injections of GH into aging mice increase total thymocyte numbers and the percentage of CD3-bearing cells, as well as the Concanavalin-A mitogenic response and IL-6 production by thymocytes. Interestingly, similar findings are observed in animals treated with IGF-1. Lastly, the thymic hypoplasia observed in dwarf mice can be reversed with GH treatment.In keeping with the data summarized earlier is the detection of receptors for PRL and GH on both thymocytes and thymic epithelial cells. Importantly, recent studies indicate that both cell types can produce PRL and GH intrathymically. Similarly, production of IGF-1 and expression of a corresponding receptor has also been demonstrated.In conclusion, these data strongly indicate that the thymus is physiologically under control of pituitary hormones PRL and GH. In addition to the classical endocrine pathway, paracrine and autocrine circuits are probably implicated in such control.

Extracellular Vesicles Isolated from Mesenchymal Stromal Cells Primed with Hypoxia: Novel strategy in Regenerative Medicine

2020 ◽  
Vol 15 ◽  
Author(s):  
Shalmali Pendse ◽  
Vaijayanti Kale ◽  
Anuradha Vaidya
Keyword(s):  
Extracellular Vesicles ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Cell Types ◽  
Molecular Profile ◽  
Cell Contact ◽  
Hematopoietic Stem

: Mesenchymal stromal cells (MSCs) regulate other cell types through a strong paracrine component called the secretome, comprising of several bioactive entities. The composition of the MSCs’ secretome is dependent upon the microenvironment in which they thrive, and hence, it could be altered by pre-conditioning the MSCs during in vitro culture. The primary aim of this review is to discuss various strategies that are being used for pre-conditioning of MSCs, also known as “priming of MSCs”, in the context of improving their therapeutic potential. Several studies have underscored the importance of extracellular vesicles (EVs) derived from primed MSCs in improving their efficacy in the treatment of various diseases. We have previously shown that co-culturing hematopoietic stem cells (HSCs) with hypoxiaprimed MSCs improves their engraftment potential. Now the question we pose is would priming of MSCs with hypoxiafavorably alter theirsecretome and would this altered secretome work as effectively as the cell to cell contact did? Here we review the current strategies of using the secretome, specifically the EVs (microvesicles and exosomes), collected from the primed MSCs with the intention of expanding HSCs ex vivo. We speculate that an effective priming of MSCs in vitrocould modulate the molecular profile of their secretome, which could eventually be used as a cell-free biologic in clinical settings.

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