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1.
BMC Musculoskelet Disord ; 22(1): 519, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090401

RESUMO

BACKGROUND: Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes. This study aimed to investigate the in vitro and in vivo protective effects of DHEA against high glucose-induced oxidative stress in tenocytes and tendons. METHODS: Tenocytes from normal Sprague-Dawley rats were cultured in low-glucose (LG) or high-glucose (HG) medium with or without DHEA. The experimental groups were: control group (LG without DHEA), LG with DHEA, HG without DHEA, and HG with DHEA. Reactive oxygen species (ROS) production, apoptosis, and messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, and interleukin-6 (IL-6) were determined. Further, diabetic rats were divided into a control group and a DHEA-injected group (DHEA group). NOX1 and NOX4 protein expression and mRNA expression of NOX1, NOX4, IL-6, matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-2, and type I and III collagens in the Achilles tendon were determined. RESULTS: In rat tenocytes, DHEA decreased the expression of NOX1 and IL-6, ROS accumulation, and apoptotic cells. In the diabetic rat Achilles tendon, NOX1 protein expression and mRNA expression of NOX1, IL-6, MMP-2, TIMP-2, and type III collagen were significantly lower while type I collagen expression was significantly higher in the DHEA group than in the control group. CONCLUSIONS: DHEA showed antioxidant and anti-inflammatory effects both in vitro and in vivo. Moreover, DHEA improved tendon matrix synthesis and turnover, which are affected by hyperglycemic conditions. DHEA is a potential preventive drug for diabetic tendinopathy.


Assuntos
Diabetes Mellitus Experimental , Tenócitos , Animais , Células Cultivadas , Desidroepiandrosterona/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Glucose/toxicidade , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley
2.
Mater Sci Eng C Mater Biol Appl ; 126: 112145, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34082956

RESUMO

Increased bone loss and risk of fracture are two of the main challenges for cancer patients who undergo ionizing radiation (IR) therapy. This decline in bone quality is in part, caused by the excessive and sustained release of reactive oxygen species (ROS). Cerium oxide nanoparticles (CeONPs) have proven antioxidant and regenerative properties and the purpose of this study was to investigate the effect of CeONPs in reducing IR-induced functional damage in human bone marrow-derived mesenchymal stromal cells (hBMSCs). hBMSCs were supplemented with CeONPs at a concentration of either 1 or 10 µg/mL 24 h prior to exposure to a single 7 Gy irradiation dose. ROS levels, cellular proliferation, morphology, senescence, DNA damage, p53 expression and autophagy were evaluated as well as alkaline phosphatase, osteogenic protein gene expression and bone matrix deposition following osteogenic differentiation. Results showed that supplementation of CeONPs at a concentration of 1 µg/mL reduced cell senescence and significantly augmented cell autophagy (p = 0.01), osteogenesis and bone matrix deposition >2-fold (p = 0.0001) while under normal, non-irradiated culture conditions. Following irradiation, functional damage was attenuated and CeONPs at both 1 or 10 µg/mL significantly reduced ROS levels (p = 0.05 and 0.001 respectively), DNA damage by >4-fold (p < 0.05) while increasing autophagy >3.5-fold and bone matrix deposition 5-fold (p = 0.0001 in both groups). When supplemented with 10 µg/mL, p53 expression increased 3.5-fold (p < 0.05). We conclude that cellular uptake of CeONPs offered a significant, multifunctional and protective effect against IR-induced cellular damage while also augmenting osteogenic differentiation and subsequent new bone deposition. The use of CeONPs holds promise as a novel multifunctional therapeutic strategy for irradiation-induced bone loss.


Assuntos
Cério , Nanopartículas , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Cério/farmacologia , Humanos , Osteogênese
3.
Mater Sci Eng C Mater Biol Appl ; 126: 112147, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34082958

RESUMO

Low proliferation capacity of corneal endothelial cells (CECs) and worldwide limitations in transplantable donor tissues reveal the critical need of a robust approach for in vitro CEC growth. However, preservation of CEC-specific phenotype with increased proliferation has been a great challenge. Here we offer a biomimetic cell substrate design, by optimizing mechanical, topographical and biochemical characteristics of materials with CEC microenvironment. We showed the surprising similarity between topographical features of white rose petals and corneal endothelium due to hexagonal cell shapes and physiologically relevant cell density (≈ 2000 cells/mm2). Polydimethylsiloxane (PDMS) substrates with replica of white rose petal topography and cornea-friendly Young's modulus (211.85 ± 74.9 kPa) were functionalized with two of the important corneal extracellular matrix (ECM) components, collagen IV (COL 4) and hyaluronic acid (HA). White rose petal patterned and COL 4 modified PDMS with optimized stiffness provided enhanced bovine CEC response with higher density monolayers and increased phenotypic marker expression. This biomimetic approach demonstrates a successful platform to improve in vitro cell substrate properties of PDMS for corneal applications, suggesting an alternative environment for CEC-based therapies, drug toxicity investigations, microfluidics and organ-on-chip applications.


Assuntos
Rosa , Animais , Bovinos , Células Cultivadas , Dimetilpolisiloxanos , Células Endoteliais , Epitélio Posterior
4.
Medicina (Kaunas) ; 57(6)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067350

RESUMO

Background andObjectives: Human bone marrow-derived mesenchymal stem cells (BMSCs) are promising sources for cell-based regenerative therapy. The purpose of the present study was to elucidate the roles of age and sex on the cellular viability and osteogenic potential of BMSCs cultured in osteogenic media. Materials and Methods: Human BMSCs were isolated and expanded from 3 age groups-20s, 30s, and 50s-from both sexes. The total number of aspirates was ten, and each subgroup had five for 20s (two females and three males), three for 30s (one female and two male), and two for 50s (one female and one male). Analyses of the cell morphology, the cell viability, the expression of the stem cell marker SSEA-4, the secretion of human vascular endothelial growth factor (VEGF), the expression of Runx2 and collagen I, the metabolic activity, and the formation of mineralization nodules were performed. Results: No significant differences were found in the cell viability of human BMSCs cultured in osteogenic media among the different age groups. There were no significant differences in the expression of SSEA among the age groups or between males and females. There were no significant differences in the secretion of human VEGF between males and females. No significant differences in Runx2 or collagen I expression were noted by age or gender. Moreover, no significant differences were shown in osteogenesis by alizarin red staining. Conclusions: The human BMSCs showed no age-related decreases in cellular viability or osteogenic differentiation potential.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Medula Óssea , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Masculino , Fator A de Crescimento do Endotélio Vascular
5.
Biosensors (Basel) ; 11(5)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069382

RESUMO

The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-flow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a functional drug testing platform through the sustained maintenance of model cells.


Assuntos
Reatores Biológicos , Hepatócitos/citologia , Sobrevivência Celular , Células Cultivadas , Desenvolvimento de Medicamentos/métodos , Preparações Farmacêuticas
6.
Mater Sci Eng C Mater Biol Appl ; 126: 112131, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34082948

RESUMO

Investigating axonal myelination by Schwann cells (SCs) is crucial for understanding mechanisms underlying demyelination and remyelination, which may help gain insights into incurable disorders like neurodegenerative diseases. In this study, a gelatin-based hydrogel, gelatin methacryloyl (GelMA), was optimized to achieve the biocompatibility, porosity, mechanical stability, and degradability needed to provide high cell viability for dorsal root ganglia (DRG) neurons and SCs, and to enable their long-term coculture needed for myelination studies. The results of cell viability, neurite elongation, SC function and maturation, SC-axon interaction, and myelination were compared with two other commonly used substrates, namely collagen and Poly-d Lysine (PDL). The tuned GelMA constructs (Young's modulus of 32.6 ± 1.9 kPa and the median value of pore size of 10.3 µm) enhanced single axon generation (unlike collagen) and promoted the interaction of DRG neurons and SCs (unlike PDL). While DRG cells exhibited relatively higher viability on PDL after 48 h, i.e., 83.8%, the cells had similar survival rate on GelMA and collagen substrates, 66.7% and 61.5%, respectively. Further adjusting the hydrogel properties to achieve two distinct ranges of relatively small and large pores supported SCs to extend their processes freely and enabled physical contact with and wrapping around their corresponding axons. Staining the cells with myelin basic protein (MBA) and myelin-associated glycoprotein (MAG) revealed enhanced myelination on GelMA hydrogel compared to PDL and collagen. Moreover, the engineered porosity enhanced DRGs and SCs attachments and flexibility of movement across the substrate. This engineered hydrogel structure can now be further explored to model demyelination in neurodegenerative diseases, as well as to study the effects of various compounds on myelin regeneration.


Assuntos
Gelatina , Hidrogéis , Neurônios , Animais , Células Cultivadas , Colágeno , Gânglios Espinais , Bainha de Mielina , Ratos Sprague-Dawley , Células de Schwann
7.
Mater Sci Eng C Mater Biol Appl ; 126: 112193, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34082990

RESUMO

Mesenchymal cells (MSCs) are an attractive option as seed cells for bioprinting. However, loss of stemness and undesired differentiation reduces their effectiveness. In this study, 12 nm bioactive nanoparticles (BNPs) which could release silicon (Si) ions were used to enhance the properties of alginate/gelatin hydrogel bioink to maintain MSC stemness. By specifically leveraging biochemical signals of BNPs, bioink with defined stiffness towards osteogenic and adipogenic potential, independent of pore structure, were designed by incorporating with different concentration of BNPs. These bioink were characterized by printability, mechanical and rheological properties as well as osteogenic and adipogenic potentials. Notably, the effect of 2% BNPs addition in alginate/gelatin hydrogel on MSC stemness maintenance was confirmed by the expression of stemness markers. At higher concentrations of BNPs (5%), printability was impacted by the gelling process. We further confirmed the enhanced stemness maintenance by sweat gland lineage commitment of bioprinted MSCs in vitro. Overall, our study proved that alginate/gelatin hydrogel bioink reinforced by BNPs in the optimal concentrations could retain MSC stemness as well as support MSC growth and prolong the desired differentiation. These findings may provide a new approach to achieve the ideal therapeutic potential of MSCs in 3D bioprinting application.


Assuntos
Bioimpressão , Células-Tronco Mesenquimais , Nanopartículas , Alginatos , Animais , Células Cultivadas , Gelatina , Hidrogéis , Camundongos , Impressão Tridimensional , Engenharia Tecidual , Tecidos Suporte
8.
Nat Commun ; 12(1): 3213, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050141

RESUMO

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.


Assuntos
Fatores Quimiotáticos/metabolismo , Proteínas de Insetos/metabolismo , Leishmaniose Cutânea/imunologia , Neutrófilos/imunologia , Proteínas e Peptídeos Salivares/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Cães , Feminino , Voluntários Saudáveis , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Insetos Vetores/imunologia , Insetos Vetores/metabolismo , Insetos Vetores/parasitologia , Leishmania major/imunologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/transmissão , Masculino , Camundongos , Pessoa de Meia-Idade , Infiltração de Neutrófilos/imunologia , Cultura Primária de Células , Psychodidae/imunologia , Psychodidae/metabolismo , Psychodidae/parasitologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/isolamento & purificação , Adulto Jovem
9.
Nat Commun ; 12(1): 3222, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050150

RESUMO

Existing computational methods that use single-cell RNA-sequencing (scRNA-seq) for cell fate prediction do not model how cells evolve stochastically and in physical time, nor can they predict how differentiation trajectories are altered by proposed interventions. We introduce PRESCIENT (Potential eneRgy undErlying Single Cell gradIENTs), a generative modeling framework that learns an underlying differentiation landscape from time-series scRNA-seq data. We validate PRESCIENT on an experimental lineage tracing dataset, where we show that PRESCIENT is able to predict the fate biases of progenitor cells in hematopoiesis when accounting for cell proliferation, improving upon the best-performing existing method. We demonstrate how PRESCIENT can simulate trajectories for perturbed cells, recovering the expected effects of known modulators of cell fate in hematopoiesis and pancreatic ß cell differentiation. PRESCIENT is able to accommodate complex perturbations of multiple genes, at different time points and from different starting cell populations, and is available at https://github.com/gifford-lab/prescient .


Assuntos
Diferenciação Celular/genética , Modelos Genéticos , RNA-Seq , Análise de Célula Única/métodos , Animais , Proliferação de Células/genética , Células Cultivadas , Simulação por Computador , Conjuntos de Dados como Assunto , Aprendizado Profundo , Hematopoese/genética , Humanos , Células Secretoras de Insulina/fisiologia , Camundongos , Software , Células-Tronco/fisiologia , Processos Estocásticos
10.
Gene ; 790: 145699, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-33964380

RESUMO

Progesterone (P4) is an anti-androgen compound whose role in sperm maturation and functionality remains unclear in sheep. Here, we aimed to investigate the regulation mechanism of P4 on the epididymal secretion of dihydrotestosterone (DHT). To this end, we performed enzyme-linked immunosorbent assays, immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction to detect P4 concentration as well as StAR, P450scc, and 3ß-HSD expression in sheep epididymis. Besides, cauda epithelial cells were cultured at different concentrations of P4 (10-9-10-5 g ml-1) as well as with or without the P4 receptor (PGR) inhibitor RU486 (10-7 M) or the PI3K-AKT inhibitor LY294006 (10-7 M) to explore the effect of P4 on DHT secretion and the underlying regulatory mechanism. The results showed that the caput, corpus, and cauda of sheep epididymis could synthesize P4 but had different synthesis ability. The PGR expression levels were the highest in the cauda, followed by the corpus. In vitro cell culture showed that P4 inhibition of DHT secretion and 5α-reductase 1 and 2 expression in epididymal epithelial cells could be moderately mitigated by RU486 but not by LY294002. Our results indicated that the paracrine and autocrine P4 could affect the secretion of DHT in epididymal cells through PGR. Overall, this study provides new data regarding the involvement of P4 in sperm maturation and functionality in sheep.


Assuntos
Di-Hidrotestosterona/metabolismo , Epididimo/metabolismo , Progesterona/farmacologia , Animais , Células Cultivadas , Epididimo/efeitos dos fármacos , Feminino , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ovinos
11.
Nat Commun ; 12(1): 2813, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001876

RESUMO

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.


Assuntos
Membrana Celular/metabolismo , Lipidômica/métodos , Fosfatidato Fosfatase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Silenciamento de Genes , Homeostase/genética , Interações Hospedeiro-Parasita , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fosfatidato Fosfatase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/ultraestrutura
12.
Nat Commun ; 12(1): 2860, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001878

RESUMO

Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.


Assuntos
Regeneração Óssea/fisiologia , Calo Ósseo/fisiologia , Consolidação da Fratura/fisiologia , Fraturas Ósseas/fisiopatologia , Células-Tronco Mesenquimais/fisiologia , Músculo Esquelético/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Osteogênese/fisiologia
13.
Nat Commun ; 12(1): 2876, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001883

RESUMO

Activation of non-shivering thermogenesis is considered a promising approach to lower body weight in obesity. p62 deficiency in adipocytes reduces systemic energy expenditure but its role in sustaining mitochondrial function and thermogenesis remains unresolved. NBR1 shares a remarkable structural similarity with p62 and can interact with p62 through their respective PB1 domains. However, the physiological relevance of NBR1 in metabolism, as compared to that of p62, was not clear. Here we show that whole-body and adipocyte-specific ablation of NBR1 reverts the obesity phenotype induced by p62 deficiency by restoring global energy expenditure and thermogenesis in brown adipose tissue. Impaired adrenergic-induced browning of p62-deficient adipocytes is rescued by NBR1 inactivation, unveiling a negative role of NBR1 in thermogenesis under conditions of p62 loss. We demonstrate that upon p62 inactivation, NBR1 represses the activity of PPARγ, establishing an unexplored p62/NBR1-mediated paradigm in adipocyte thermogenesis that is critical for the control of obesity.


Assuntos
Adipócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , PPAR gama/metabolismo , Proteína Sequestossoma-1/deficiência , Termogênese , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Animais Recém-Nascidos , Núcleo Celular/metabolismo , Células Cultivadas , Metabolismo Energético/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , PPAR gama/genética , Ligação Proteica , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Proteína Sequestossoma-1/genética
14.
Nat Commun ; 12(1): 2885, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001887

RESUMO

Despite the widespread observations on the osteogenic effects of magnesium ion (Mg2+), the diverse roles of Mg2+ during bone healing have not been systematically dissected. Here, we reveal a previously unknown, biphasic mode of action of Mg2+ in bone repair. During the early inflammation phase, Mg2+ contributes to an upregulated expression of transient receptor potential cation channel member 7 (TRPM7), and a TRPM7-dependent influx of Mg2+ in the monocyte-macrophage lineage, resulting in the cleavage and nuclear accumulation of TRPM7-cleaved kinase fragments (M7CKs). This then triggers the phosphorylation of Histone H3 at serine 10, in a TRPM7-dependent manner at the promoters of inflammatory cytokines, leading to the formation of a pro-osteogenic immune microenvironment. In the later remodeling phase, however, the continued exposure of Mg2+ not only lead to the over-activation of NF-κB signaling in macrophages and increased number of osteoclastic-like cells but also decelerates bone maturation through the suppression of hydroxyapatite precipitation. Thus, the negative effects of Mg2+ on osteogenesis can override the initial pro-osteogenic benefits of Mg2+. Taken together, this study establishes a paradigm shift in the understanding of the diverse and multifaceted roles of Mg2+ in bone healing.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Macrófagos/metabolismo , Magnésio/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Fêmur/metabolismo , Fêmur/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Magnésio/administração & dosagem , Magnésio/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Proteínas Serina-Treonina Quinases/genética , Ratos Sprague-Dawley , Células THP-1 , Canais de Cátion TRPM/genética
15.
Nat Commun ; 12(1): 2856, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001893

RESUMO

Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.


Assuntos
Neutrófilos/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Ontologia Genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Peritonite/genética , Peritonite/patologia , Pneumonia/genética , Pneumonia/patologia , Baço/citologia , Baço/metabolismo
16.
Nat Commun ; 12(1): 2863, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001904

RESUMO

During injury, monocytes are recruited from the circulation to inflamed tissues and differentiate locally into mature macrophages, with prior reports showing that cavity macrophages of the peritoneum and pericardium invade deeply into the respective organs to promote repair. Here we report a dual recombinase-mediated genetic system designed to trace cavity macrophages in vivo by intersectional detection of two characteristic markers. Lineage tracing with this method shows accumulation of cavity macrophages during lung and liver injury on the surface of visceral organs without penetration into the parenchyma. Additional data suggest that these peritoneal or pleural cavity macrophages do not contribute to tissue repair and regeneration. Our in vivo genetic targeting approach thus provides a reliable method to identify and characterize cavity macrophages during their development and in tissue repair and regeneration, and distinguishes these cells from other lineages.


Assuntos
Fígado/fisiopatologia , Lesão Pulmonar/fisiopatologia , Macrófagos/fisiologia , Monócitos/fisiologia , Cavidade Peritoneal/fisiologia , Cavidade Pleural/fisiologia , Animais , Linhagem da Célula/genética , Células Cultivadas , Fígado/lesões , Ativação de Macrófagos/fisiologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Monócitos/citologia , Monócitos/metabolismo , Cavidade Peritoneal/citologia , Fagocitose/fisiologia , Cavidade Pleural/citologia
17.
Nat Commun ; 12(1): 2875, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001908

RESUMO

Polymeric drug carriers are widely used for providing temporal and/or spatial control of drug delivery, with corticosteroids being one class of drugs that have benefitted from their use for the treatment of inflammatory-mediated conditions. However, these polymer-based systems often have limited drug-loading capacity, suboptimal release kinetics, and/or promote adverse inflammatory responses. This manuscript investigates and describes a strategy for achieving controlled delivery of corticosteroids, based on a discovery that low molecular weight corticosteroid dimers can be processed into drug delivery implant materials using a broad range of established fabrication methods, without the use of polymers or excipients. These implants undergo surface erosion, achieving tightly controlled and reproducible drug release kinetics in vitro. As an example, when used as ocular implants in rats, a dexamethasone dimer implant is shown to effectively inhibit inflammation induced by lipopolysaccharide. In a rabbit model, dexamethasone dimer intravitreal implants demonstrate predictable pharmacokinetics and significantly extend drug release duration and efficacy (>6 months) compared to a leading commercial polymeric dexamethasone-releasing implant.


Assuntos
Corticosteroides/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Dexametasona/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Corticosteroides/química , Corticosteroides/farmacocinética , Animais , Células Cultivadas , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Dexametasona/química , Dexametasona/farmacocinética , Dimerização , Modelos Animais de Doenças , Implantes de Medicamento , Liberação Controlada de Fármacos , Polímeros/química , Coelhos , Ratos , Uveíte/metabolismo , Uveíte/prevenção & controle
18.
Nat Commun ; 12(1): 3006, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021143

RESUMO

Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity.


Assuntos
/imunologia , Inflamação/imunologia , Ativação Linfocitária/imunologia , Pneumonia/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Basófilos/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Neutrófilos/imunologia , Adulto Jovem
19.
Virulence ; 12(1): 1111-1121, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34034617

RESUMO

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.


Assuntos
Células Epiteliais/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Coronavirus Respiratório Porcino/fisiologia , Interferência Viral , Animais , Antígenos CD13/metabolismo , Células Cultivadas , Coinfecção/virologia , Infecções por Coronavirus/virologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Imunidade Inata , Infecções por Orthomyxoviridae/virologia , Suínos , Traqueia/citologia , Replicação Viral
20.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 35(5): 611-619, 2021 May 15.
Artigo em Chinês | MEDLINE | ID: mdl-33998216

RESUMO

Objective: To investigate the effect of silk fibroin-poly- L-lactic acid (SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells (ADSCs). Methods: ADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers (set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group (group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells. Results: ADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar ( P>0.05). qRT-PCR results showed that chondrogenesis related genes (aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes (peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences ( P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs. Conclusion: The ADSCs can be amplified by SF-PLLA microcarriers, and the chondrogenic differential ability of harvested cells was up-regulated while the adipogenic differential was down-regulated.


Assuntos
Tecido Adiposo/citologia , Fibroínas , Ácido Láctico , Células-Tronco/citologia , Diferenciação Celular , Células Cultivadas , Humanos
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