scholarly journals Platelet Derived Extracellular Vesicles Promote in Vitro Gingival Wound Healing.

2021 ◽  
Author(s):  
Miquel Antich-Rosselló ◽  
Marta Munar-Bestard ◽  
Maria Antònia Forteza-Genestra ◽  
Javier Calvo ◽  
Antoni Gayà ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Matrix Remodeling ◽  
Platelet Concentrates ◽  
Ldh Activity ◽  
Exclusion Chromatography ◽  
Good Biocompatibility

Abstract Purpose: Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates like platelet lysate (PL). In this article, the use of extracellular vesicles (EVs) derived from PL and their combination with hyaluronic acid biomaterials (HA) in wound healing was investigated.Methods: EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro for biocompatibility (LDH activity and metabolic activity) and by a wound healing assay and gene expression analysis.Results: EVs and EVs-HA treatments were biocompatible and showed an increase in wound healing compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed in gingival keratinocytes and gingival fibroblasts after the treatment with EVs.Conclusion: EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet derived EVs emerge as promising candidates for oral regeneration with the possibility to combine them with biomaterials in order to enhance their application in clinical use.

scholarly journals Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use

2020 ◽  
Author(s):  
R.M.S. Cardoso ◽  
S.C. Rodrigues ◽  
C. F. Gomes ◽  
F.V. Duarte ◽  
M. Romao ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Extracellular Vesicles ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Size Exclusion ◽  
Matrix Remodeling

ABSTRACTExtracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and up-scalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content.Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and up-scaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate skin regeneration in a mouse model of delayed wound healing.The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and up-scalability required for mass production of sEV to be used in a clinical setting.

scholarly journals Exosomal tau with seeding activity is released from Alzheimer’s disease synapses, and seeding potential is associated with amyloid beta

2021 ◽  
Author(s):  
Emily Miyoshi ◽  
Tina Bilousova ◽  
Mikhail Melnik ◽  
Danyl Fakhrutdinov ◽  
Wayne W. Poon ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Human Pathology ◽  
Exclusion Chromatography ◽  
Flow Through ◽  
Aggregation Size ◽  
Synaptic Vesicle Release ◽  
Induced Aggregation

AbstractSynaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2–10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aβ release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aβ42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aβ. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aβ within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.

Characterization and regulation of extracellular vesicles in the lumen of the ovine uterus†

2020 ◽  
Vol 102 (5) ◽  
pp. 1020-1032 ◽  
Author(s):  
Eleanore V O’Neil ◽  
Gregory W Burns ◽  
Christina R Ferreira ◽  
Thomas E Spencer
Keyword(s):  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Rna Seq ◽  
Interferon Tau ◽  
Uterine Lumen ◽  
Pregnant Sheep ◽  
Exclusion Chromatography ◽  
Ovine Uterus

Abstract Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.

67 Optimizing a protocol for isolating extracellular vesicles from medium conditioned by bovine embryos in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 159
Author(s):  
K. C. Pavani ◽  
A. Hendrix ◽  
B. Leemans ◽  
A. Van Soom
Keyword(s):  
Conditioned Medium ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Downstream Processing ◽  
Size Exclusion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Isolation Method ◽  
Exclusion Chromatography

In the absence of the maternal tract, pre-implantation bovine embryos cultured in group are able to promote their own development in vitro by releasing autocrine embryotropins. Recently we have identified extracellular vesicles (EV) among these embryotropins as one of the communication mechanisms among embryos. Extracellular vesicles are nano-sized (25-250nm), with a lipid bilayer, and are functionally active, since they contain proteins, lipids, and nucleic acids, including RNA and miRNA. However, one of the major challenges in isolating EV is an inadequate volume of medium conditioned by bovine embryo. As it requires larger volumes of conditioned medium to isolate EV, our study mainly focused on isolating high yields of functional EV from a minimal volume. There are 3 known isolation methods for EV: differential ultracentrifugation (DU), OptiPrep™ density gradient ultracentrifugation (ODGU), and size-exclusion chromatography (SEC). We have used these 3 protocols to determine the method that yielded the highest number of EV. We used routine in vitro maturation and fertilization methods, but for in vitro culture presumed zygotes were cultured until 8 days post-insemination (dpi) in medium (synthetic oviducal fluid supplemented with insulin, transferrin, selenium, and bovine serum albumin) that was ultracentrifuged to remove any possible contaminating EV. In vitro embryo culture took place in groups of 25 presumed zygotes in 50-mL drops, covered with mineral oil and incubated at 38°C in 5% CO2, 5% O2, and 90% N2. On 8 dpi, medium conditioned by bovine embryo was collected and pooled until 3mL. For each isolation method, 1mL of conditioned medium was used, and next, EV isolated from each isolation method were analysed with nanoparticle tracking, electron microscopy, and Western blot (CD9, Flotillin 1, and AGO 2). We observed higher concentrations (1.03×109 particles mL−1) of EV were isolated from the SEC compared with the other 2 methods (301.5×108 particles mL−1 and 64.5×108 particles mL−1 for DU and ODGU, respectively; P<0.05), whereas smaller size EV (20-50nm) were lost during the ultracentrifugation methods. Besides, it takes only 2h of time to perform size-exclusion chromatography for isolating EV, whereas it takes more than 1 day to perform ultracentrifugation methods. Therefore, we propose to use SEC for further downstream processing and sequencing of miRNA in isolated EV. We are currently focusing on optimizing an EV isolation protocol to extract EV from very low volumes of conditioned medium (less than 500 µL).

scholarly journals Small Extracellular Vesicles from adipose derived stromal cells significantly attenuate in vitro the NF-κB dependent inflammatory/catabolic environment of osteoarthritis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Carola Cavallo ◽  
Giulia Merli ◽  
Rosa Maria Borzì ◽  
Nicoletta Zini ◽  
Stefania D’Adamo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Nuclear Translocation ◽  
Protein Release ◽  
Biologically Active ◽  
Size Exclusion ◽  
Chromatography Method ◽  
Adipose Derived Stromal Cells

AbstractThe therapeutic ability of Mesenchymal Stem/Stromal Cells to address osteoarthritis (OA) is mainly related to the secretion of biologically active factors, which can be found within their secreted Extracellular Vesicles including small Extracellular Vesicles (sEV). Aim of this study was to investigate the effects of sEV from adipose derived stromal cells (ADSC) on both chondrocytes and synoviocytes, in order to gain insights into the mechanisms modulating the inflammatory/catabolic OA environment. sEV, obtained by a combined precipitation and size exclusion chromatography method, were quantified and characterized, and administered to chondrocytes and synoviocytes stimulated with IL-1β. Cellular uptake of sEV was evaluated from 1 to 12 h. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h, when p65 nuclear translocation was investigated to study NF-κB pathway. This study underlined the potential of ADSC derived sEV to affect gene expression and protein release of both chondrocytes and synoviocytes, counteracting IL-1β induced inflammatory effects, and provided insights into their mechanisms of action. sEV uptake was faster in synoviocytes, where it also elicited stronger effects, especially in terms of cytokine and chemokine modulation. The inflammatory/catabolic environment mediated by NF-κB pathway was significantly attenuated by sEV, which hold promise as new therapeutic strategy to address OA.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

scholarly journals A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 157
Author(s):  
Kinga Böszörményi ◽  
Janet Hirsch ◽  
Gwendoline Kiemenyi Kayere ◽  
Zahra Fagrouch ◽  
Nicole Heijmans ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Neutralizing Antibodies ◽  
Size Exclusion ◽  
Vitro Assay ◽  
Usutu Virus ◽  
Transmission Electron ◽  
Exclusion Chromatography ◽  
Efficacious Vaccine ◽  
Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 522
Author(s):  
Elmo W. I. Neuberger ◽  
Barlo Hillen ◽  
Katharina Mayr ◽  
Perikles Simon ◽  
Eva-Maria Krämer-Albers ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Affinity Capture ◽  
Healthy Humans ◽  
Dnase Digestion ◽  
And Topology ◽  
Single Bout ◽  
Exclusion Chromatography ◽  
Free Circulating Dna ◽  
Line Elements

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

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