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1.
Nanoscale ; 13(30): 13158, 2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34477799

RESUMO

Correction for 'Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes' by Fabio Perissinotto et al., Nanoscale, 2021, 13, 5224-5233, DOI: .


Assuntos
Vesículas Extracelulares , Membrana Celular
2.
Nanoscale ; 13(10): 5224-5233, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33687046

RESUMO

Extracellular vesicles (EVs) are a potent intercellular communication system. Such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite their extreme biological relevance, their mechanisms of interaction with the membranes of recipient cells are still hotly debated. Here, we propose a multiscale investigation based on atomic force microscopy, small angle X-ray scattering, small angle neutron scattering and neutron reflectometry to reveal structure-function correlations of purified EVs in interaction with model membrane systems of variable complex compositions and to spot the role of different membrane phases on the vesicle internalization routes. Our analysis reveals strong interactions of EVs with the model membranes and preferentially with the borders of protruding phase domains. Moreover, we found that upon vesicle breaking on the model membrane surface, the biomolecules carried by/on EVs diffuse with different kinetics rates, in a process distinct from simple fusion. The biophysical platform proposed here has clear implications on the modulation of EV internalization routes by targeting specific domains at the plasma cell membrane and, as a consequence, on EV-based therapies.


Assuntos
Vesículas Extracelulares , Comunicação Celular , Membrana Celular , Microscopia de Força Atômica
3.
Clin Transl Med ; 10(8): e262, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33377658

RESUMO

The lack of approved anti-inflammatory and neuroprotective therapies in otology has been acknowledged in the last decades and recent approaches are heralding a new era in the field. Extracellular vesicles (EVs) derived from human multipotent (mesenchymal) stromal cells (MSC) can be enriched in vesicular secretome fractions, which have been shown to exert effects (eg, neuroprotection and immunomodulation) of their parental cells. Hence, MSC-derived EVs may serve as novel drug candidates for several inner ear diseases. Here, we provide first evidence of a strong neuroprotective potential of human stromal cell-derived EVs on inner ear physiology. In vitro, MSC-EV preparations exerted immunomodulatory activity on T cells and microglial cells. Moreover, local application of MSC-EVs to the inner ear significantly attenuated hearing loss and protected auditory hair cells from noise-induced trauma in vivo. Thus, EVs derived from the vesicular secretome of human MSC may represent a next-generation biological drug that can exert protective therapeutic effects in a complex and nonregenerating organ like the inner ear.

4.
Front Neurol ; 10: 1225, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849808

RESUMO

Spinal cord injury is characterized by initial neural tissue disruption that triggers secondary damage and extensive non-resolving inflammation, which aggravates loss of function and hinders recovery. The early onset of inflammation following traumatic spinal cord injury underscores the importance of acute intervention after the initial trauma. Injections of mesenchymal stromal cells (MSCs) can reduce inflammation following spinal cord injury. We asked if extracellular vesicles (EVs) can substitute the anti-inflammatory and anti-scarring activities of their parental MSCs in a rat model of contusion spinal cord injury. We report that MSC-EVs were as potent as the parental intact cells in reducing the level of neuroinflammation for up to 2 weeks post-injury. Acute application of EVs after spinal cord injury was shown to robustly decrease the expression of pro-inflammatory cytokines in the spinal cord parenchyma in the very early phase of secondary damage. Moreover, the anti-scarring impact of MSC-EVs was even more efficient than the parental cells. We therefore conclude that anti-inflammatory and anti-scarring activities of MSC application can be mediated by their secreted EVs. In light of their substantial safety and druggability advantages, EVs may have a high potential in early therapeutic treatment following traumatic spinal cord injury.

5.
Sci Rep ; 9(1): 7258, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076619

RESUMO

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.


Assuntos
Expressão Gênica/fisiologia , Heparina/metabolismo , Células Estromais/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Soro/metabolismo , Transdução de Sinais/fisiologia , Cordão Umbilical/metabolismo
6.
Cytotherapy ; 21(6): 581-592, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30979664

RESUMO

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) may deliver therapeutic effects that are comparable to their parental cells. MSC-EVs are promising agents for the treatment of a variety of diseases. To reach the intermediate goal of clinically testing safety and efficacy of EVs, strategies should strive for efficient translation of current EV research. On the basis of our in vitro an in vivo findings regarding the biological actions of EVs and our experience in manufacturing biological stem cell therapeutics for routine use and clinical testing, we discuss strategies of manufacturing and quality control of umbilical cord-derived MSC-EVs. We introduce guidelines of good manufacturing practice and their practicability along the path from the laboratory to the patient. We present aspects of manufacturing and final product quality testing and highlight the principle of "The process is the product." The approach presented in this perspective article may facilitate translational research during the development of complex biological EV-based therapeutics in a very early stage of manufacturing as well as during early clinical safety and proof-of-concept testing.


Assuntos
Técnicas de Cultura de Células/métodos , Vesículas Extracelulares/fisiologia , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Animais , Produtos Biológicos/normas , Produtos Biológicos/uso terapêutico , Técnicas de Cultura de Células/normas , Ensaios Clínicos como Assunto , Estabilidade de Medicamentos , Humanos , Controle de Qualidade
7.
Int J Mol Sci ; 18(7)2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28671586

RESUMO

The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.


Assuntos
Vesículas Extracelulares/metabolismo , Imunomodulação , Células Estromais/metabolismo , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Exossomos/imunologia , Exossomos/metabolismo , Vesículas Extracelulares/imunologia , Humanos , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
8.
Int J Mol Sci ; 18(6)2017 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-28587212

RESUMO

Extracellular vesicles (EVs) derived from stem and progenitor cells may have therapeutic effects comparable to their parental cells and are considered promising agents for the treatment of a variety of diseases. To this end, strategies must be designed to successfully translate EV research and to develop safe and efficacious therapies, whilst taking into account the applicable regulations. Here, we discuss the requirements for manufacturing, safety, and efficacy testing of EVs along their path from the laboratory to the patient. Development of EV-therapeutics is influenced by the source cell types and the target diseases. In this article, we express our view based on our experience in manufacturing biological therapeutics for routine use or clinical testing, and focus on strategies for advancing mesenchymal stromal cell (MSC)-derived EV-based therapies. We also discuss the rationale for testing MSC-EVs in selected diseases with an unmet clinical need such as critical size bone defects, epidermolysis bullosa and spinal cord injury. While the scientific community, pharmaceutical companies and clinicians are at the point of entering into clinical trials for testing the therapeutic potential of various EV-based products, the identification of the mode of action underlying the suggested potency in each therapeutic approach remains a major challenge to the translational path.


Assuntos
Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Fracionamento Celular/métodos , Vesículas Extracelulares , Animais , Fracionamento Celular/normas , Micropartículas Derivadas de Células , Exossomos , Humanos
9.
Cytotherapy ; 19(4): 458-472, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28188071

RESUMO

BACKGROUND AIMS: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs. METHODS: Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. RESULTS: Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. CONCLUSIONS: The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.


Assuntos
Técnicas de Cultura de Células/normas , Engenharia Celular/normas , Vesículas Extracelulares/transplante , Indústria Manufatureira/normas , Células-Tronco Mesenquimais/citologia , Adipogenia/efeitos dos fármacos , Adipogenia/fisiologia , Diferenciação Celular/efeitos dos fármacos , Engenharia Celular/métodos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Indústria Manufatureira/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/ultraestrutura , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Guias de Prática Clínica como Assunto/normas , Padrões de Referência
10.
Emerg Infect Dis ; 20(12): 2119-22, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25418009

RESUMO

West Nile virus (WNV) is becoming more widespread and markedly effecting public health. We sequenced the complete polyprotein gene of a divergent WNV strain newly detected in a pool of Uranotaenia unguiculata mosquitoes in Austria. Phylogenetic analyses suggest that the new strain constitutes a ninth WNV lineage or a sublineage of WNV lineage 4.


Assuntos
Culicidae/virologia , Vírus do Nilo Ocidental/classificação , Animais , Áustria , Feminino , Genes Virais , Dados de Sequência Molecular , Filogenia , RNA Viral , Febre do Nilo Ocidental/transmissão
11.
Infect Genet Evol ; 27: 96-104, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25017655

RESUMO

Batai virus (BATV) is a poorly studied arthropod-borne virus belonging to the genus Orthobunyavirus (Bunyamwera serogroup) within the family Bunyaviridae. It has been associated with human influenza-like febrile illness in several Asian, African, and European countries. Calovo virus (CVOV), isolated in 1960 in Slovakia, has been classified as BATV based on high antigenic similarity, and since then both CVOV and BATV were used as synonyms. In order to fully clarify the phylogenetic relationships between CVOV, BATV, and other members of the Bunyamwera serogroup, we performed whole genome sequencing of four CVOV strains isolated in Europe and phylogenetic analyses of all related viruses. The nucleocapsid protein, encoded by the S genomic segment, contains 233 amino acids, 60 of which, putatively critical for protein function, are conserved. Within the CVOV polyprotein encoded by the M genomic segment, putative cleavage sites, N-glycosylation sites, and seven transmembrane regions were identified. The RNA-dependent RNA polymerase, encoded by the L genome segment, exhibits conservation of the three regions known to be conserved among bunyavirus and arenavirus L proteins. Phylogenetic analyses of all three genomic segments of selected orthobunyaviruses clearly revealed that European and Asian/African strains of BATV are phylogenetically different and form two distinct lineages, indicating the existence of two different genotypes of BATV, tentatively named European genotype (with CVOV as a type strain) and Afro-Asian genotype (with BATV as a type strain) of BATV.


Assuntos
Genoma Viral , Genômica , Orthobunyavirus/classificação , Orthobunyavirus/genética , Evolução Molecular , Humanos , Dados de Sequência Molecular , Filogenia , RNA Viral , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sorogrupo , Proteínas Virais/genética
12.
Infect Genet Evol ; 20: 124-30, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23988729

RESUMO

Ilesha virus is an arthropod-borne virus belonging to the genus Orthobunyavirus of the Bunyaviridae family. Ilesha virus has been isolated from humans in several African countries, mostly in relation with febrile illness and erythema, though there are reported cases of fatal meningoencephalitis and hemorrhagic fever. In the present study, we report the complete genomic sequence of all three Ilesha virus segments (S, M, L) and characterize the open reading frames. The nucleoprotein encoded by segment S contains 59 conserved orthobunyavirus amino acids putatively critical for protein function. For the polyprotein encoded by segment M, potential proteolytic cleavage sites and N-glycosylation sites as well as conserved cysteines are described in reference to other orthobunyaviruses. Within the C terminal glycoprotein Gc a putative fusion peptide could be localized. In the RNA-dependent RNA polymerase encoded by segment L, all strictly conserved amino acids within the four conserved regions known to be catalytically active are present. Phylogenetic analyses conducted for each Ilesha virus genomic segment confirm the classification of Ilesha virus within the Bunyamwera serogroup of orthobunyaviruses. Ilesha virus segments S and L exhibit highest genetic conservation with Bunyamwera virus and Ngari virus, with maximum sequence identities of 88% for segment S and 82% for segment L. However, the M segment was found to be more diverse with a maximum nucleotide identity of 72% to Bunyamwera serogroup viruses.


Assuntos
Infecções por Bunyaviridae/virologia , Genoma Viral/genética , Orthobunyavirus/genética , África , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Orthobunyavirus/classificação , Orthobunyavirus/isolamento & purificação , Filogenia , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas Virais de Fusão/genética
13.
J Gen Virol ; 94(Pt 11): 2449-2457, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23939978

RESUMO

Barkedji virus, named after the area of its first identification in Senegal, is a newly discovered flavivirus (FV), for which we propose the abbreviation BJV. In the present study, we report the first-time detection of BJV in Culex perexiguus mosquitoes in Israel in 2011 and determination of its almost complete polyprotein gene sequence. We characterized the BJV genome and defined putative mature proteins, conserved structural elements and potential enzyme motifs along the polyprotein precursor. By comparing polyproteins and individual proteins of BJV with several other FVs, a distant relationship of BJV to Nounane virus (NOUV), a recently described African FV, is demonstrated. Phylogenetic analysis of 55 selected flaviviral polyprotein gene sequences exhibits two major clusters, one made up of the classical three clades of FVs: mosquito-borne, tick-borne and those without known vectors. The other cluster exclusively contains so-called 'insect-specific' FVs, which do not replicate in vertebrate cells. Based on our phylogenetic analysis, BJV is related to other members of the mosquito-borne clade with yet unknown vertebrate hosts, such as NOUV, Donggang virus, Chaoyang virus and Lammi virus. However, with a maximum identity of only 54 % to NOUV, BJV represents a distinct new virus species.


Assuntos
Culex/virologia , Flavivirus/classificação , Flavivirus/isolamento & purificação , Poliproteínas/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Flavivirus/genética , Genoma Viral , Israel , Dados de Sequência Molecular , Filogenia , Poliproteínas/química , Senegal , Especificidade da Espécie , Proteínas Virais/química , Proteínas Virais/genética
14.
Virol J ; 8: 48, 2011 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-21288362

RESUMO

The nonstructural proteins 1 (NS1) from influenza A and B viruses are known as the main viral factors antagonising the cellular interferon (IFN) response, inter alia by inhibiting the retinoic acid-inducible gene I (RIG-I) signalling. The cytosolic pattern-recognition receptor RIG-I senses double-stranded RNA and 5'-triphosphate RNA produced during RNA virus infections. Binding to these ligands activates RIG-I and in turn the IFN signalling. We now report that the influenza C virus NS1 protein also inhibits the RIG-I-mediated IFN signalling. Employing luciferase-reporter assays, we show that expression of NS1-C proteins of virus strains C/JJ/50 and C/JHB/1/66 considerably reduced the IFN-ß promoter activity. Mapping of the regions from NS1-C of both strains involved in IFN-ß promoter inhibition showed that the N-terminal 49 amino acids are dispensable, while the C-terminus is required for proper modulation of the IFN response. When a mutant RIG-I, which is constitutively active without ligand binding, was employed, NS1-C still inhibited the downstream signalling, indicating that IFN inhibitory properties of NS1-C are not necessarily linked to an RNA binding mechanism.


Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , Evasão da Resposta Imune , Influenzavirus C/patogenicidade , Interferon beta/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Bioensaio , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Genes Reporter , Humanos , Influenzavirus C/imunologia , Interferon beta/biossíntese , Luciferases/genética , Luciferases/metabolismo , Mapeamento de Interação de Proteínas
15.
J Mol Genet Med ; 4: 239-46, 2010 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-20838663

RESUMO

We report the establishment of a reverse-genetics system for the rescue of recombinant influenza C/JJ/50 virus from seven plasmids. The nucleotide sequence of the whole C/JJ/50 genome was determined and full-length cDNAs were cloned into an RNA pol I/pol II-based bidirectional vector. Transfection of Vero cells and subsequent amplification on MDCK cells yielded viral HA titres of 128. The utility of this bidirectional approach was proved by generating a reassortant virus encoding the NS segment from strain C/JHB/1/66 and a virus with mutations in the noncoding ends of PB1. The latter virus, which has a base-pair mutation within the proposed double-stranded region of the PB1 termini, exhibited impaired replication. In conclusion, our efficient seven-plasmid system for the rescue of recombinant influenza C virus may be used to study the influenza C virus life cycle in more detail and for generation of influenza C virus-based vectors.

16.
FEMS Yeast Res ; 5(3): 271-80, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15556089

RESUMO

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.


Assuntos
Proteína S6 Ribossômica/fisiologia , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia , Leveduras/fisiologia , Sequência de Bases , DNA Fúngico/química , DNA Fúngico/genética , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína Ribossômica L10 , Proteína S6 Ribossômica/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Leveduras/genética
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