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1.
PLoS Biol ; 18(8): e3000807, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760056

RESUMO

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.


Assuntos
Células da Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Proteínas de Ligação a DNA/genética , Inflamassomos/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Ligação a Fosfato/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas de Ligação a DNA/deficiência , Feminino , Fêmur/citologia , Fêmur/metabolismo , Regulação da Expressão Gênica , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteínas de Ligação a Fosfato/deficiência , Piroptose/genética , Piroptose/efeitos da radiação , Transdução de Sinais , Baço/metabolismo , Baço/patologia , Baço/efeitos da radiação , Transplante Isogênico , Irradiação Corporal Total , Raios X
2.
Int J Nanomedicine ; 15: 3903-3920, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606657

RESUMO

Background: Researchers are trying to study the mechanism of neural stem cells (NSCs) differentiation to oligodendrocyte-like cells (OLCs) as well as to enhance the selective differentiation of NSCs to oligodendrocytes. However, the limitation in nerve tissue accessibility to isolate the NSCs as well as their differentiation toward oligodendrocytes is still challenging. Purpose: In the present study, a hybrid polycaprolactone (PCL)-gelatin nanofiber scaffold mimicking the native extracellular matrix and axon morphology to direct the differentiation of bone marrow-derived NSCs to OLCs was introduced. Materials and Methods: In order to achieve a sustained release of T3, this factor was encapsulated within chitosan nanoparticles and chitosan-loaded T3 was incorporated within PCL nanofibers. Polyaniline graphene (PAG) nanocomposite was incorporated within gelatin nanofibers to endow the scaffold with conductive properties, which resemble the conductive behavior of axons. Biodegradation, water contact angle measurements, and scanning electron microscopy (SEM) observations as well as conductivity tests were used to evaluate the properties of the prepared scaffold. The concentration of PAG and T3-loaded chitosan NPs in nanofibers were optimized by examining the proliferation of cultured bone marrow-derived mesenchymal stem cells (BMSCs) on the scaffolds. The differentiation of BMSCs-derived NSCs cultured on the fabricated scaffolds into OLCs was analyzed by evaluating the expression of oligodendrocyte markers using immunofluorescence (ICC), RT-PCR and flowcytometric assays. Results: Incorporating 2% PAG proved to have superior cell support and proliferation while guaranteeing electrical conductivity of 10.8 × 10-5 S/cm. Moreover, the scaffold containing 2% of T3-loaded chitosan NPs was considered to be the most biocompatible samples. Result of ICC, RT-PCR and flow cytometry showed high expression of O4, Olig2, platelet-derived growth factor receptor-alpha (PDGFR-α), O1, myelin/oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) high expressed but low expression of glial fibrillary acidic protein (GFAP). Conclusion: Considering surface topography, biocompatibility, electrical conductivity and gene expression, the hybrid PCL/gelatin scaffold with the controlled release of T3 may be considered as a promising candidate to be used as an in vitro model to study patient-derived oligodendrocytes by isolating patient's BMSCs in pathological conditions such as diseases or injuries. Moreover, the resulted oligodendrocytes can be used as a desirable source for transplanting in patients.


Assuntos
Materiais Biomiméticos/farmacologia , Células da Medula Óssea/citologia , Diferenciação Celular , Nanofibras/química , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Tecidos Suporte/química , Compostos de Anilina/química , Animais , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica , Gelatina/química , Grafite/química , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/ultraestrutura , Células-Tronco Neurais/metabolismo , Oligodendroglia/efeitos dos fármacos , Poliésteres/química , Ratos , Suínos , Tri-Iodotironina/farmacologia
3.
DNA Cell Biol ; 39(9): 1691-1699, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32700968

RESUMO

Long noncoding RNAs (lncRNAs) contribute toward regulating gene expression and cell differentiation and may be involved in the pathogenesis of several diseases. The objective of this study was to determine the expression patterns of lncRNAs in bone marrow mesenchymal stem cells (BMSCs) derived from patients with osteoporotic fractures and their relevance to osteogenic function. The BMSCs were isolated from the femoral head of patients with hip fractures (FRX) and controls with osteoarthritis (OA). We found 74 differentially expressed genes between FRX and OA, of which 33 were of the lncRNA type. Among them, 52 genes (20 lncRNAs) were replicated in another independent dataset. The differentially expressed lncRNAs were over-represented among those correlated with differentially expressed protein-coding genes. In addition, the comparison of pre- and post-differentiated paired samples revealed 163 differentially expressed genes, of which 99 were of the lncRNA type. Among them, the overexpression of LINC00341 induced an upregulation of typical osteoblastic genes. In conclusion, the analysis of lncRNA expression in BMSCs shows specific patterns in patients with osteoporotic fractures, as well as changes associated with osteogenic differentiation. The regulation of bone genes through lncRNAs might bring new opportunities for designing bone anabolic therapies in systemic and localized bone disorders.


Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoporose/genética , RNA Longo não Codificante/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Células HEK293 , Humanos , Células-Tronco Mesenquimais/citologia , Osteogênese , Osteoporose/patologia , RNA Longo não Codificante/genética , Transcriptoma
4.
PLoS One ; 15(6): e0233773, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559198

RESUMO

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived ß-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not ß-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1ß, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than ß-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both ß-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives.


Assuntos
Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Fatores Imunológicos/farmacologia , Probióticos/farmacologia , Suínos/imunologia , beta-Glucanas/farmacologia , Ração Animal , Animais , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Escherichia coli , Fatores Imunológicos/administração & dosagem , Interleucinas/genética , Interleucinas/metabolismo , Probióticos/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , beta-Glucanas/administração & dosagem
5.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(6): 781-786, 2020 Jun 15.
Artigo em Chinês | MEDLINE | ID: mdl-32538572

RESUMO

Objective: To investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: hBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot. Results: Compared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased ( P<0.05); the above indexes in group D were significantly decreased ( P<0.05); the above indexes between groups C, E and group A were not significantly different ( P>0.05). Conclusion: miR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.


Assuntos
Células da Medula Óssea , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Osteogênese/genética
6.
Stem Cells Dev ; 29(12): 747-754, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: covidwho-209923

RESUMO

This prospective nonrandomized open-label cohort study addresses the safety and efficacy of exosomes (ExoFlo™) derived from allogeneic bone marrow mesenchymal stem cells as treatment for severe COVID-19. During April 2020, ExoFlo was provided to 24 SARS-CoV-2 polymerase chain reaction-positive patients at a single hospital center, all of whom met criteria for severe COVID-19 as well as moderate-to-severe acute respiratory distress syndrome. Patients received a single 15 mL intravenous dose of ExoFlo and were evaluated for both safety and efficacy from days 1 to 14 post-treatment. All safety endpoints were met with no adverse events observed within 72 h of ExoFlo administration. A survival rate of 83% was observed. In total, 17 of 24 (71%) patients recovered, 3 of 24 (13%) patients remained critically ill though stable, and 4 of 24 (16%) patients expired for reasons unrelated to the treatment. Overall, after one treatment, patients' clinical status and oxygenation improved with an average pressure of arterial oxygen to fraction of inspired oxygen ratio (PaO2/FiO2) increase of 192% (P < 0.001). Laboratory values revealed significant improvements in absolute neutrophil count [mean reduction 32% (P value <0.001)] and lymphopenia with average CD3+, CD4+, and CD8+ lymphocyte counts increasing by 46% (P < 0.05), 45% (P < 0.05), and 46% (P < 0.001), respectively. Likewise, acute phase reactants declined, with mean C-reactive protein, ferritin, and D-dimer reduction of 77% (P < 0.001), 43% (P < 0.001), and 42% (P < 0.05), respectively. In conclusion, owing to its safety profile, capacity to restore oxygenation, downregulate cytokine storm, and reconstitute immunity, ExoFlo is a promising therapeutic candidate for severe COVID-19. Future randomized controlled trials (RCTs) are needed to determine ExoFlo therapeutic potential.


Assuntos
Células da Medula Óssea/citologia , Infecções por Coronavirus/terapia , Estado Terminal/terapia , Exossomos/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Pneumonia Viral/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/patogenicidade , Micropartículas Derivadas de Células/transplante , Estudos de Coortes , Infecções por Coronavirus/mortalidade , Estado Terminal/mortalidade , Feminino , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/ultraestrutura , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/mortalidade , Índice de Gravidade de Doença , Resultado do Tratamento
7.
Nat Commun ; 11(1): 2570, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444631

RESUMO

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.


Assuntos
Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Switching de Imunoglobulina , Memória Imunológica , Baço/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Animais Selvagens/imunologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea/citologia , Ciclo Celular , Proliferação de Células/genética , Regulação da Expressão Gênica/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Baço/citologia , Células Estromais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
8.
Stem Cells Dev ; 29(12): 747-754, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32380908

RESUMO

This prospective nonrandomized open-label cohort study addresses the safety and efficacy of exosomes (ExoFlo™) derived from allogeneic bone marrow mesenchymal stem cells as treatment for severe COVID-19. During April 2020, ExoFlo was provided to 24 SARS-CoV-2 polymerase chain reaction-positive patients at a single hospital center, all of whom met criteria for severe COVID-19 as well as moderate-to-severe acute respiratory distress syndrome. Patients received a single 15 mL intravenous dose of ExoFlo and were evaluated for both safety and efficacy from days 1 to 14 post-treatment. All safety endpoints were met with no adverse events observed within 72 h of ExoFlo administration. A survival rate of 83% was observed. In total, 17 of 24 (71%) patients recovered, 3 of 24 (13%) patients remained critically ill though stable, and 4 of 24 (16%) patients expired for reasons unrelated to the treatment. Overall, after one treatment, patients' clinical status and oxygenation improved with an average pressure of arterial oxygen to fraction of inspired oxygen ratio (PaO2/FiO2) increase of 192% (P < 0.001). Laboratory values revealed significant improvements in absolute neutrophil count [mean reduction 32% (P value <0.001)] and lymphopenia with average CD3+, CD4+, and CD8+ lymphocyte counts increasing by 46% (P < 0.05), 45% (P < 0.05), and 46% (P < 0.001), respectively. Likewise, acute phase reactants declined, with mean C-reactive protein, ferritin, and D-dimer reduction of 77% (P < 0.001), 43% (P < 0.001), and 42% (P < 0.05), respectively. In conclusion, owing to its safety profile, capacity to restore oxygenation, downregulate cytokine storm, and reconstitute immunity, ExoFlo is a promising therapeutic candidate for severe COVID-19. Future randomized controlled trials (RCTs) are needed to determine ExoFlo therapeutic potential.


Assuntos
Células da Medula Óssea/citologia , Infecções por Coronavirus/terapia , Estado Terminal/terapia , Exossomos/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Pneumonia Viral/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/patogenicidade , Micropartículas Derivadas de Células/transplante , Estudos de Coortes , Infecções por Coronavirus/mortalidade , Estado Terminal/mortalidade , Feminino , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/ultraestrutura , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/mortalidade , Índice de Gravidade de Doença , Resultado do Tratamento
9.
PLoS One ; 15(5): e0228510, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407317

RESUMO

Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (≥75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.


Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Células-Tronco Adultas/fisiologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Linhagem da Célula/genética , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Neurônios/fisiologia
10.
Nat Commun ; 11(1): 2280, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385245

RESUMO

Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.


Assuntos
Células da Medula Óssea/citologia , Linhagem da Célula , Rim/embriologia , Macrófagos/citologia , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/sangue , Quimiocina CX3CL1/metabolismo , Feminino , Feto/citologia , Fígado/embriologia , Masculino , Camundongos , Monócitos/citologia
11.
Nat Commun ; 11(1): 1747, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269263

RESUMO

Receptor interacting protein kinase 1 (RIPK1) regulates cell death and inflammatory responses downstream of TNFR1 and other receptors, and has been implicated in the pathogenesis of inflammatory and degenerative diseases. RIPK1 kinase activity induces apoptosis and necroptosis, however the mechanisms and phosphorylation events regulating RIPK1-dependent cell death signaling remain poorly understood. Here we show that RIPK1 autophosphorylation at serine 166 plays a critical role for the activation of RIPK1 kinase-dependent apoptosis and necroptosis. Moreover, we show that S166 phosphorylation is required for RIPK1 kinase-dependent pathogenesis of inflammatory pathologies in vivo in four relevant mouse models. Mechanistically, we provide evidence that trans autophosphorylation at S166 modulates RIPK1 kinase activation but is not by itself sufficient to induce cell death. These results show that S166 autophosphorylation licenses RIPK1 kinase activity to induce downstream cell death signaling and inflammation, suggesting that S166 phosphorylation can serve as a reliable biomarker for RIPK1 kinase-dependent pathologies.


Assuntos
Apoptose , Inflamação/metabolismo , Inflamação/patologia , Fosfosserina/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Alanina Transaminase/metabolismo , Animais , Células da Medula Óssea/citologia , Colite/patologia , Genótipo , Hepatite/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Neoplasias/patologia , Fosforilação , Sepse/patologia , Pele/patologia , Fator de Necrose Tumoral alfa
12.
Nat Commun ; 11(1): 1817, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286311

RESUMO

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1's role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.


Assuntos
Apresentação do Antígeno/imunologia , Células Dendríticas/metabolismo , Endossomos/metabolismo , Cinesina/metabolismo , Ácidos/metabolismo , Animais , Antígenos/metabolismo , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Proliferação de Células , Endocitose , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinesina/deficiência , Camundongos Knockout , Camundongos Transgênicos , Microtúbulos/metabolismo , Neoplasias/patologia , Ovalbumina/imunologia , Solubilidade
13.
Nat Biomed Eng ; 4(5): 518-530, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32313101

RESUMO

The detection and quantification of low-abundance molecular biomarkers in biological samples is challenging. Here, we show that a plasmonic nanoscale construct serving as an 'add-on' label for a broad range of bioassays improves their signal-to-noise ratio and dynamic range without altering their workflow and readout devices. The plasmonic construct consists of a bovine serum albumin scaffold with approximately 210 IRDye 800CW fluorophores (with a fluorescence intensity approximately 6,700-fold that of a single 800CW fluorophore), a polymer-coated gold nanorod acting as a plasmonic antenna and biotin as a high-affinity biorecognition element. Its emission wavelength can be tuned over the visible and near-infrared spectral regions by modifying its size, shape and composition. It improves the limit of detection in fluorescence-linked immunosorbent assays by up to 4,750-fold and is compatible with multiplexed bead-based immunoassays, immunomicroarrays, flow cytometry and immunocytochemistry methods, and it shortens overall assay times (to 20 min) and lowers sample volumes, as shown for the detection of a pro-inflammatory cytokine in mouse interstitial fluid and of urinary biomarkers in patient samples.


Assuntos
Bioensaio/métodos , Corantes Fluorescentes/química , Nanopartículas/química , Animais , Células da Medula Óssea/citologia , Linhagem Celular Tumoral , Coloides/química , Células Dendríticas/citologia , Feminino , Citometria de Fluxo , Fluorescência , Humanos , Imunoensaio , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Microesferas , Proteômica , Padrões de Referência
14.
Nat Commun ; 11(1): 2054, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345968

RESUMO

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.


Assuntos
Células Dendríticas/citologia , Nicho de Células-Tronco , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CXCL12/farmacologia , Análise por Conglomerados , Colágeno/farmacologia , Células Dendríticas/efeitos dos fármacos , Combinação de Medicamentos , Humanos , Laminina/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteoglicanas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
15.
Sheng Li Xue Bao ; 72(2): 167-174, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32328610

RESUMO

Humans with chronic psychological stress are prone to develop multiple disorders of body function including impairment of immune system. Chronic psychological stress has been reported to have negative effects on body immune system. However, the underlying mechanisms have not been clearly demonstrated. All immune cells are derived from hematopoietic stem cells (HSC) in the bone marrow, including myeloid cells which comprise the innate immunity as a pivotal component. In this study, to explore the effects of chronic psychological stress on HSC and myeloid cells, different repeated restraint sessions were applied, including long-term mild restraint in which mice were individually subjected to a 2 h restraint session twice daily (morning and afternoon/between 9:00 and 17:00) for 4 weeks, and short-term vigorous restraint in which mice were individually subjected to a 16 h restraint session (from 17:00 to 9:00 next day) for 5 days. At the end of restraint, mice were sacrificed and the total cell numbers in the bone marrow and peripheral blood were measured by cell counting. The proportions and absolute numbers of HSC (Lin-CD117+Sca1+CD150+CD48-) and myeloid cells (CD11b+Ly6C+) were detected by fluorescence activated cell sorting (FACS) analysis. Proliferation of HSC was measured by BrdU incorporation assay. The results indicated that the absolute number of HSC was increased upon long-term mild restraint, but was decreased upon short-term vigorous restraint with impaired proliferation. Both long-term mild restraint and short-term vigorous restraint led to the accumulation of CD11b+Ly6C+ cells in the bone marrow as well as in the peripheral blood, as indicated by the absolute cell numbers. Taken together, long-term chronic stress led to increased ratio and absolute number of HSC in mice, while short-term stress had opposite effects, which suggests that stress-induced accumulation of CD11b+Ly6C+ myeloid cells might not result from increased number of HSC.


Assuntos
Proliferação de Células , Células-Tronco Hematopoéticas/citologia , Restrição Física , Estresse Psicológico , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/citologia , Antígeno CD11b/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
16.
Adv Exp Med Biol ; 1254: 1-22, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32323265

RESUMO

Since the identification of B cells in 1965 (Cooper  et al. 1965), three has been tremendous progress in our understanding of B cell development, maturation and function. A number of B cell subpopulations, including B-1, B-2 and regulatory B cells, have been identified. B-1 cells mainly originate from the fetal liver and contain B-1a and B-1b subsets. B-2 cells are derived from the bone marrow (BM) and can be further classified into follicular B (FOB) and marginal zone B (MZB) cells. Regulatory B cells (Bregs) function to suppress immune responses, primarily by production of the anti-inflammatory cytokine IL-10. B cell tolerance is established at several checkpoints, during B cell development in the BM (central tolerance) as well as during B cell maturation and activation in the periphery (peripheral tolerance). This chapter will focus on the regulation of important processes during the development and maturation of B-1 and B-2 cells.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Tolerância Imunológica , Ativação Linfocitária , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Humanos , Tolerância Periférica
17.
PLoS One ; 15(4): e0230507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255777

RESUMO

The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.


Assuntos
Plaquetas/metabolismo , Células da Medula Óssea/citologia , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Suínos , Trombina/farmacologia
18.
Braz J Med Biol Res ; 53(4): e9282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267311

RESUMO

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Células Dendríticas/citologia , Receptores de Glutamato Metabotrópico/fisiologia , Células Th17/imunologia , Vitiligo/imunologia , Animais , Citometria de Fluxo , Masculino , Melaninas/biossíntese , Melanócitos/citologia , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/imunologia , Células Th17/citologia , Vitiligo/genética
19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(2): 145-151, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32314712

RESUMO

Objective To construct the hematopoietic microenvironment system simulating different hematopoietic sites during the embryonic stages for in vitro inducing the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hematopoietic cells, and to initially evaluate the efficiency of differentiation. Methods Stereomicroscopy and HE staining were used to observe the anatomical parts and morphology of yolk sac (YS), placenta (PL) and fetal liver (FL) of E11.5 mice. The embryonic YS, PL and FL tissues from the stages of E7.5-E9.5, E10.5-E12.5 and E13.5-E15.5 were collected for stromal cell culture, and conditioned culture media were prepared, namely, yolk sac stromal cell conditioned medium (YSSC-CM), placental SC-CM (PLSC-CM) and fetal liver SC-CM (FLSC-CM), and co-cultured with in vitro expanded SD rat BMSCs. The experimental cells were divided into control group, interleukin 6(IL-6) combined with stem cell factor (SCF) treatment group, YSSC-CM treatment group, PLSC-CM treatment group and FLSC-CM treatment group. After co-culture for 7-9 days, the floating cells in culture medium were collected. Giemsa staining was used to exam cell morphology. Direct immunofluorescence was conducted to detect CD34 and CD45 expression. Colony formation assay was performed to detect granulocyte/macrophage colony formation unit (GM-CFU) to identify differentiated cells. Results Under a stereomicroscope, PL and FL of mouse embryos had the same position as human embryos, while YS was wrapped on the outer side of embryo body and amniotic membrane. HE staining showed that PL vessel labyrinth, FL sinusoids and YS blood islands were rich in blood cells. The inverted phase-contrast microscope and cell counting results indicated that the floating cells in the culture medium of the YSSC-CM group, the PLSC-CM group and the FLSC-CM group significantly increased compared with the control group and the IL-6 combined with SCF group, especially the FLSC-CM group had the most. The floating cells in YSSC-CM group, PLSC-CM group and FLSC-CM group were similar in morphology to lymphoid or mononucleoid cells as showed by Giemsa staining, and expressed hematopoietic cell-specific surface markers CD34 and CD45. The number of hematopoietic cell colonies formed was the most in the FLSC-CM group, followed by the PLSC-CM group, and the least in the YSSC-CM group. The control group and IL-6 combined with SCF treatment group had no changes. Conclusion By collecting YS, PL, and FL according to the stage and time sequence, YSSC-CM, PLSC-CM, and FLSC-CM prepared can induce the differentiation of SD rat's BMSCs into hematopoietic cells, and FLSC-CM-treated cells have better differentiation efficiency.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Fígado , Camundongos , Placenta , Gravidez , Ratos , Ratos Sprague-Dawley , Saco Vitelino
20.
Int J Nanomedicine ; 15: 497-511, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158207

RESUMO

Introduction: RNA-based therapy for bone repair and regeneration is a highly safe and effective approach, which has been extensively investigated in recent years. However, the molecular stability of RNA agents still remains insufficient for clinical application. High porosity, tunable size, and ideal biodegradability and biosafety are a few of the characters of mesoporous silicon nanoparticles (MSNs) that render them a promising biomaterial carrier for RNA treatment. Materials and Methods: In this study, a novel miR-26a delivery system was constructed based on MSNs. Next, we assessed the miRNA protection of the delivery vehicles. Then, rat bone marrow mesenchymal stem cells (rBMSCs) were incubated with the vectors, and the transfection efficiency, cellular uptake, and effects on cell viability and osteogenic differentiation were evaluated. Results: The results demonstrated that the vectors protected miR-26a from degradation in vitro and delivered it into the cytoplasm. A relatively low concentration of the delivery systems significantly increased osteogenic differentiation of rBMSCs. Conclusion: The vectors constructed in our study provide new methods and strategies for the delivery of microRNAs in bone tissue engineering.


Assuntos
Diferenciação Celular , Técnicas de Transferência de Genes , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Nanopartículas/química , Osteogênese/genética , Animais , Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Iminas/química , Células-Tronco Mesenquimais/fisiologia , Peptídeos/química , Polietilenos/química , Porosidade , Ratos Sprague-Dawley , Dióxido de Silício/química , Transfecção
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